Principles of Bioenergetics
123
Vladimir P. Skulachev
Belozersky Institute of Physico-Chemical
Biology
Moscow State University
Moscow
Russia
Felix O. Kasparinsky
Faculty of Biology
Moscow State University
Moscow
Russia
Alexander V. Bogachev
Belozersky Institute of Physico-Chemical
Biology
Moscow State University
Moscow
Russia
ISBN 978-3-642-33429-0
DOI 10.1007/978-3-642-33430-6
ISBN 978-3-642-33430-6
(eBook)
Preface
Energy and life. These are the phenomena of objective reality, subjective notions,
and simply the words of our language. It has been already over 2000 years that
philosophers have been arguing about the essence of these terms. Scientists have
been trying to comprehend them for centuries. It has been already for many
decades that the positive emotional impact of these words has been used in the
advertising industry to attract consumers attention. Millions of people use the
words energy and life every day while being satisfied with only an illusion of
intuitive understanding of their meaning.
What is the secret of such popularity? We would like to suggest that evolution
of human language has been determined by the success of those individuals who
could attract wide attention to the notions that proved to be the most essential for
the very existence of the society as a whole and its individual representatives.
Advantages of civilization allow modern people to avoid many problems that
previous generations had to face. This makes it possible to change the system of
priorities. Energy and life seem to be not of such a crucial importance today
both for an individual and human society in general. Emotional attractiveness
encoded in the language can be viewed today as a historical step on the path that
was taken by people so as to understand Nature around them as well as themselves.
The years 19611995 witnessed achievement of unbelievable progress in understanding of the molecular mechanisms of energy supply. This fact could create the
illusion that practically everything has already been discovered in , and the next
generations of biologists would better choose some other areas of scientific
research.
But with the arrival of year 1996 there appeared the first publications on a
completely new role of mitochondriaenergy transforming organelles of animal,
plant, and fungal cellsin the destiny of these cells. Mitochondria were found to
play a key role in the processes leading to programmed cell death. They are not
only the main energy providers for this process (and this fact leads to a new
approach to the problem of energy and death that was previously viewed mainly
in connection to nuclear weapons), but also serve as extremely powerful facilitators of lethal signals. Hence there appeared a new area of research connected to
v
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Preface
the role of mitochondria in the programmed elimination of tissue parts, organs, and
even the whole organisms. This discovery led to a substantial increase in the
number of scientific publications on mitochondria-related issues. The number of
such articles in the first decade of the twenty-first century was about 2.5 times
higher than in the last decade of the previous century.
We hope this book will become the contemporary textbook on bioenergetics.
Most of it is based on the course of lectures on that has been presented by one of
the books authors (V. P. S.) to Moscow State University students over the last
40 years. This course embraces fundamental data on molecular mechanisms of
accumulation of energy and its usage in mitochondrial, chloroplast, and bacterial
membranes. These issues have already been reviewed in the previous textbook by
the first author (Skulachev VP (1988) Membrane Bioenergetics. Springer, Berlin),
but the present book includes also a number of new aspects, e.g. evolution of
bioenergetic mechanisms, toxicology and physiology of reactive oxygen species,
their role in programmed death phenomena, such as apoptosis, mitoptosis and
phenoptosis (aging), as well as some other topics.
Contents
Part I
1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1 Definition of the Term Bioenergetics and Some
Milestones of its History . . . . . . . . . . . . . . . . . . . . .
1.2 Bioenergetics in the System of Biological Sciences . .
1.3 Laws of Bioenergetics. . . . . . . . . . . . . . . . . . . . . . .
1.4 Evolution of Bioenergetic Mechanisms . . . . . . . . . . .
1.4.1 Adenosine Triphosphate . . . . . . . . . . . . . . .
1.4.2 Hypothesis of Adenine-Based Photosynthesis.
1.4.3 Reserve Energy Sources and Glycolysis . . . .
1.4.4 Proton Channels and H+-ATPase as Means
to Prevent Glycolysis-Induced Acidification
of the Cell . . . . . . . . . . . . . . . . . . . . . . . . .
1.4.5 Bacteriorhodopsin-Based Photosynthesis
as the Primordial Mechanism of Visible
Light Energy Transduction . . . . . . . . . . . . .
1.4.6 Chlorophyll-Based Photosynthesis . . . . . . . .
1.4.7 Respiratory Mechanism of Energy Supply . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Part II
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Contents
Ways to Use D
lH Generated by the Cyclic
Photoredox Chain . . . . . . . . . . . . . . . . . . .
2.2 Noncyclic Photoredox Chain of Green Bacteria . . . .
2.3 Noncyclic Photoredox Chain of Chloroplasts
and Cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . .
2.3.1 Principle of Functioning . . . . . . . . . . . . . .
2.3.2 Photosystem 1 . . . . . . . . . . . . . . . . . . . . .
2.3.3 Photosystem 2 . . . . . . . . . . . . . . . . . . . . .
2.3.4 Cytochrome b6f Complex . . . . . . . . . . . . .
2.3.5 Fate of D
lH Generated by the Chloroplast
Photosynthetic Redox Chain . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
Organotrophic Energetics . . . . . . . . . . . . . . . .
3.1 Substrates of Organotrophic Energetics . . .
3.2 Short Review of Carbohydrate Metabolism
3.3 Mechanism of Substrate Phosphorylation. .
3.4 Energetic Efficiency of Fermentation . . . .
3.5 Carnosine . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Contents
Part III
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D
lH Consumers
D
lH -Driven Chemical Work . . . . . . . . . . . . . . . . . . . . . .
7.1 H+-ATP Synthase . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1.1 Subunit Composition of H+-ATP Synthase . . .
7.1.2 Three-Dimensional Structure and Arrangement
in the Membrane . . . . . . . . . . . . . . . . . . . . .
7.1.3 ATP hydrolysis by Isolated Factor F1 . . . . . . .
7.1.4 Synthesis of Bound ATP by Isolated Factor F1
7.1.5 Fo-Mediated H+Conductance . . . . . . . . . . . . .
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Contents
7.1.6
D
lH -Driven Mechanical Work: Bacterial Motility . . . . . .
8.1 D
lH Powers the Flagellar Motor . . . . . . . . . . . . . . . .
8.2 Structure of the Bacterial Flagellar Motor . . . . . . . . . .
8.3 A Possible Mechanism of the H+-motor . . . . . . . . . . .
8.4 D
lH -Driven Movement of Non-Flagellar Motile
Prokaryotes and Intracellular Organelles of Eukaryotes .
8.5 Motile Eukaryote: Prokaryote Symbionts. . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D
lH -Driven Osmotic Work . . . . . . . . . . . . . . . . . . . .
9.1 Definition and Classification . . . . . . . . . . . . . . . .
9.2 DW As Driving Force . . . . . . . . . . . . . . . . . . . . .
9.3 DpH As Driving Force . . . . . . . . . . . . . . . . . . . .
9.4 Total D
lH as Driving Force . . . . . . . . . . . . . . . .
9.5 D
lH -Driven Transport Cascades . . . . . . . . . . . . .
9.6 Carnitine: An Example of a Transmembrane Group
9.7 Some Examples of D
lH -Driven Carriers . . . . . . .
9.7.1 Escherichia coli Lactose, H+-Symporter . .
9.7.2 Mitochondrial ATP/ADP-Antiporter . . . . .
9.8 Role of D
lH in Transport of Macromolecules. . . .
9.8.1 Transport of Mitochondrial Proteins:
Biogenesis of Mitochondria . . . . . . . . . . .
9.8.2 Transport of Bacterial Proteins . . . . . . . . .
9.8.3 Role of DW in Protein Arrangement
in the Membrane . . . . . . . . . . . . . . . . . .
9.8.4 Bacterial DNA Transport. . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Contents
xi
10 D
lH as Energy Source for Heat Production . . . . . . . . . . . . .
10.1 Three Ways of Converting Metabolic Energy into Heat . .
10.2 Thermoregulatory Activation of Free
Respiration in Animals . . . . . . . . . . . . . . . . . . . . . . . . .
10.2.1 Brown Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.2.2 Skeletal Muscles. . . . . . . . . . . . . . . . . . . . . . . .
10.3 Thermoregulatory Activation of Free Respiration in Plants
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Part IV
Part V
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12 D
lNa Generators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.1 Na+-Motive Decarboxylases. . . . . . . . . . . . . . . . . . . . . . .
12.2 Na+-Translocating NADH:Quinone-Oxidoreductase . . . . . .
12.2.1 Primary Structure of Subunits of Na+-Translocating
NADH:Quinone Oxidoreductase . . . . . . . . . . . . . .
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Contents
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13 Utilization of D
lNa Produced by Primary D
lNa Generators . .
13.1 Osmotic Work Supported by D
lNa . . . . . . . . . . . . . . . . .
13.1.1 Na+, Metabolite-Symporters . . . . . . . . . . . . . . . . .
13.1.2 Na+ Ions and Regulation of Cytoplasmic pH . . . . .
13.2 Mechanical Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.3 Chemical Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.3.1 D
lNa -Driven ATP Synthesis in Anaerobic Bacteria .
13.3.2 D
lNa Consumers Performing Chemical Work
in Methanogenic Archaea . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Part VI
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Contents
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15.2.5 Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15.2.6 Necrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15.2.7 Phenoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15.3 Biological Function of ROS. . . . . . . . . . . . . . . . . . . . . . . .
15.4 Aging as Slow Phenoptosis Caused by Increase
in mROS Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15.4.1 Definition of the Term Aging and a Short
Historical Overview of the Problem . . . . . . . . . . . .
15.4.2 Phenoptosis of Organisms that Reproduce
Only Once . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15.4.3 Can Aging be a Slow Form of Phenoptosis? . . . . . .
15.4.4 Mutations that Prolong Lifespan . . . . . . . . . . . . . . .
15.4.5 ROS and Aging . . . . . . . . . . . . . . . . . . . . . . . . . .
15.4.6 Naked Mole-Rat . . . . . . . . . . . . . . . . . . . . . . . . . .
15.4.7 Aging Program: Working Hypothesis . . . . . . . . . . .
15.4.8 Paradox of Protein p53 . . . . . . . . . . . . . . . . . . . . .
15.4.9 Arrest of Age-Dependent Increase of Mitochondrial
ROS as a Possible Way to Slow
the Aging Program . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Contents
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Index of Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abbreviations
DW
D
lH
D
lNa
DpH
ADP
AMP
AOX
ATP
BLM
BChl
(BChl)2
(BChl)*2
(BChl)+
2
BPheo
cAMP
Capsaicin
CCCP
Chl
(Chl)2
(Chl)*2
(Chl)+
2
CoQ
CoQH2
CoQH
CoQEm
EPR
FAD
FMN
xvi
Fd
GDP
GTP
HQNO
H+/e
MitoQ
MQ
NAC
NAD+
NADH
NADP+
NADPH
Na+-NQR
Na+/e
NDH-1
NDH-2
NMR
PC
Pheo
Pi
PQ
PQH2
PQPPi
ROS
SkQ
SkQ1
SkQR1
TNF
Abbreviations
Ferredoxin
Guanosine 50 -diphosphate
Guanosine 50 -triphosphate
2-Heptyl-4-hydroxyquinoline N-oxide
Ratio of protons to electrons transferred across the membrane
by respiratory or photosynthetic chain enzyme complexes
10-(60 -ubiquinonyl)decyltriphenylphosphonium
Menaquinone
N-Acetylcysteine
Nicotinamide adenine dinucleotide
Reduced nicotinamide adenine dinucleotide
Nicotinamide adenine dinucleotide phosphate
Reduced nicotinamide adenine dinucleotide phosphate
Na+-translocating NADH:ubiquinone-oxidoreductase
Ratio of sodium ions to electrons transferred across the membrane
by a respiratory chain enzyme
Bacterial H+-translocating NADH:quinone-oxidoreductase, homologue of mitochondrial complex I
Bacterial noncoupled NADH:quinone-oxidoreductase
Nuclear magnetic resonance
Plastocyanin
Pheophytin
Inorganic phosphate
Plastoquinone
Plastoquinol
Plastosemiquinone anion
Inorganic pyrophosphate
Reactive oxygen species
Plastoquinone derivative bound to delocalized cation
through decyl or amyl linker
10-(60 -Plastoquinonyl)decyltriphenylphosphonium
10-(60 -Plastoquinonyl)decylrhodamine
Tumor necrosis factor
Part I
Chapter 1
Introduction
1 Introduction
1.1 Definition of the Term Bioenergetics and Some Milestones of its History
1 Introduction
more general to more specific matters. The top storey is for biosphere studies. The
second storey from the top is occupied by ecology. It deals with the communities
of individual organisms that form ecosystems that in turn constitute the biosphere
when brought together. The next storey deals with the group of sciences that can
be united under the title biology of species. These are the classical descriptive
biological sciences such as zoology, botany, mycology, microbiology, and
virology.
The species-composing populations are in turn composed of individuals who
are studied at the fourth storey from the top. Here specialists in anatomy and
physiology study the structure and functioning of individual organisms and their
organs and tissues. Tissues are composed of cells, and biological phenomena
studied on this level from the realm of cellular biology (the fifth storey from the
top). The next level of simplification is subcellular biology, which studies intracellular organelles and supramolecular systems. This realm includes studies on
structure and functioning of plasmalemma, cytosol, nucleus, mitochondria,
endoplasmic reticulum, lysosomes, peroxisomes, chloroplasts, and some other
subcellular organelles.
The seventh storey from the top is of crucial importance. It is occupied by
molecular biology. Individual molecules that compose living beingsespecially
macromolecules and their complexesare the domain of molecular biology. It is
on this storey that the transition to quantitative description of biological objects
occurs. The biology of molecules is a very complex, but at the same time it is a
science that operates on a level of precision similar to that of chemistry and
physics. Molecules and their complexes that are the objects of research here must
be kept in their native condition, i.e., they need to maintain their biological
function when being studied. When we move to the lowest storey (eighth from the
top) this condition is not necessarily observed. Bioorganic chemistry studies the
structural and physicochemical properties of pure individual macromolecules as
well as their constituents and low molecular mass compounds present in living
organisms. Some of these compounds are inorganic, but it makes little sense to
consider bioinorganic chemistry as a special science and make a separate storey
for it, since the number of its objects is very much smaller than that of bioorganic
chemistry.
The classification of the biological sciences is not limited to the levels of
complexity. A biological object can be studied by a biologist, chemist, physicist,
or mathematician. Each of these specialists uses different methodological
approaches and research philosophy. Sometimes even the conclusions of the
concrete researcher depend on the methodological porch of the biology
building he is working in. The mathematician strives to find equations that
describe the object of research or applies informatics to analyze biological texts.
The biophysicist, while studying the reactions flow over time, wants his observations to have high time resolution over the whole period of observation.1 In the
case of the chemist, it is the purity of molecules and system homogeneity that will
be of primary importance. The biologist is not likely to strive for femtosecond
definition, mathematical accuracy, or chemical purity. He is especially interested
in the living systems behavior (regulation, birth, and death), and also in its historyphylogenesis and ontogenesis. The authors of this book are biologists by
education, and that is why the book to a substantial degree reflects the biological
approach.
There is yet other criterion for the classification of biological studiesbiological functions of the systems which are the objects of research. There are four
main functions of biological objects. One of them is genetic, connected with the
transfer of individual properties to the next generation. The science dealing with
this function is genetics. The second vital condition of a living systems existence
is energy supply, and it is bioenergetics that studies these phenomena. There is
also an important biological function consisting in interconversion of different
chemical substances. The compounds we consist of are basically different from
those that can be found in the environment. The great majority of them we synthesize ourselvesthat is why transformation of substances can be viewed as a
special biological function. By analogy with genetics and bioenergetics, the science investigating metabolism of substances can be called metabolics (Skulachev
1988). This is an example of a neologism. We basically speak here about classical
biochemistry. But at a certain point biochemistry gave birth to the sciences that
1
Russian biophysicists have been quite successful in this area. For instance, the laboratories of the
Belozersky Institute of Physico-Chemical biology at Moscow State University have measured
kinetics of the primary processes of photosynthesis in living nature to the resolution of 2 9 10-15 s,
which is the highest achieved in biological studies) (Shuvalov 2007).
1 Introduction
1:1
This principle was first stated in 1941 by Fritz Lipmann (Fig. 1.4), who at that
time knew of only one form of biological currencyATP (Lipmann 1941). It is
true that ATP is a universal sign of lifethere are no living cells without ATP. But
later two additional kinds of biological currency were found. These are electrochemical potential differences of hydrogen ions and sodium ionsD
lH and
D
lNa , respectively.
The electrochemical potential difference in H+ ions (D
lH ) can exist in two
formselectrical and chemical. The first is a transmembrane difference in electric
potential DW, and the second is the transmembrane difference in H+ concentration DpH. Figure 1.5 illustrates these two notions. If there is an electric
potential difference across the membrane (generated, e.g. by a battery), H+ tends to
move from the positively charged to the negatively charged compartment
(Fig. 1.5a). One can reach the same situation by adding, for instance, hydrochloric
acid to the left compartment, thus increasing the concentration of H+ in the left
compartment in comparison to the right one. In this case, a proton current will also
take place downhill, but it is DpH, and not DW, that will be the driving force for
the process (Fig. 1.5b).
Potential energy accumulated in the form of DW or DpH can be utilized if the
membrane has a device capable of coupling downhill H+ movement to the performance of useful work.
The energy stored in D
lH can be calculated from Eq. 1.2.
D
lH FDW RT ln
H p
H n
1:2
10
1 Introduction
the molar concentrations of H+ in the positively charged (or more acidic) and
negatively charged (or more alkaline) compartments, respectively (Mitchell 1973).
The units of D
lH are J mol-1. To express D
lH in volts (V), one should divide
it by Faradays constant (F). For this quantity, Mitchell introduced the term
proton motive force (Dp) (Mitchell 1973), which at 25 C can be calculated
according to Eq. 1.3.
Dp D
lH =F DW 0:06 DpH:
1:3
The difference between DW and DpH is due to the fact that the pH is a negative
logarithm of the H+ concentration. Indeed, D
lH increases when the left compartment in Fig. 1.5 becomes more positively charged or its pH is lowered.
According to Eq. 1.3, DpH = 1 is equivalent to DW = 0.06 V (or 60 mV). The
same value expressed in kJ mol-1 is 5.7, and that in kcal mol-1 is 1.37.
Similar equations can be applied to sodium energetics. In this case, in Eq. 1.3
D
lH is replaced by D
lNa :, and the proton motive force by the sodium ion motive
force (Eq. 1.4). The latter can be abbreviated as Ds (from Latin sodium) (Skulachev 1984, 1988).
Ds D
lNa =F DW 0:06 DpNa
11
1:4
Figure 1.6a shows a scheme that describes the energetics of living cells using
D
lH as the convertible membrane-linked energy currency. According to the
scheme, the energy of light or respiratory substrates can be utilized by enzymes of
the photosynthetic or respiratory redox chains or by bacteriorhodopsin to form
D
lH . The latter can support various types of work in the protonic membrane,
with ATP synthesis being the most important. Substrate-level phosphorylations
serve as an alternative mechanism of ATP formation that operates with no D
lH
involved. Such phosphorylations occur in the glycolytic chain and in oxidative
decarboxylation of a-ketoglutarate.
The D
lH -linked formation of ATP is a major but not the only process of
transformation of D
lH into chemical work. The D
lH -supported synthesis
of inorganic pyrophosphate and transfer of reducing equivalents in the direction of
more negative redox potentials (e.g., reverse electron transfer in the respiratory
chain and the transhydrogenase reaction) are also of this type of energy
transduction.
The D
lH -driven uphill transport of various substances across the coupling
membrane can be described as D
lH ? osmotic work transduction, while the
rotation of the protonic motor of motile bacteria represents D
lH -driven
mechanical work. Heat production by mitochondria of cold-exposed animals is an
example of D
lH ? heat production.
All the above types of energy transduction have also been described for the
nonmembranous parts of cells. Here they are supported by the energy of ATP or
other high-energy compounds.
There are systems specializing in the buffering of D
lH or ATP levels. For
D
lH , this function is performed by gradients of Na+ and K+; for ATP, by creatine
phosphate (Skulachev 1988).
In certain bacteria, D
lNa instead of D
lH is formed at the expense of energy
released by respiration or by nonoxidative decarboxylation of some organic acids.
Then the D
lNa can be used to support chemical, osmotic, or mechanical work.
The K+/H+ gradient can serve as a buffering system for D
lNa (Skulachev 1988)
(Fig. 1.6b).
The most complicated pattern of energy transduction is inherent in animal cells,
where there are three different interconvertible energy currenciesD
lH for
mitochondria and some other intracellular vesicles, D
lNa for the outer cell
membrane, and ATP for nonmembranous cell constituents.
Coming back to the first law of bioenergetics, we should state that its three
components (ATP, D
lH , and D
lNa ) do not have equal value. ATP is always
present, while the two other components are interchangeable, and hence some
organisms have only one or the other. But one of the latter two should always be
present. Thus we can formulate the second law.
12
1 Introduction
Second law of bioenergetics. Every living cell has at least two forms of interconvertible energyATP, and D
lH or D
lNa . If we continue our analogy to
money, the living cell always has both cash and a credit card in its wallet.
Third law of bioenergetics. This law states that there is interconversion of three
forms of energy stored in a cell. That is why a cell can satisfy all its energy needs
if it can obtain at least one of the three interconvertible energy forms from external
energy sources (Fig. 1.6c). In other words, it makes no difference for the cell
whether it pays with cash or with a credit card.
There are some kinds of cells that use only one type of energy source. Some of
them live by respiration or photosynthesis and produce only D
lH ; they have no
glycolysis, hence they have no possibility for direct synthesis of ATP. For some
others, vice versa, glycolysis (which forms ATP without mediation of D
lH ) is the
13
only energy source. Certain bacteria have neither glycolysis nor respiration nor
photosynthesis. They accumulate D
lNa via one of the decarboxylation reactions
and use D
lNa for production of ATP and D
lH (Skulachev 1988).
Most people are not aware of the scale of events that accompany our bodys
energy supply. Here are some of the numbers. The average human being consumes
about 140 l of oxygen per day and synthesizes about 40 kg of ATP. To obtain such
values, about 0.5 kg of protons needs to be transported across mitochondrial
membranes, while D
lH generators maintain the voltage of the electric field across
the mitochondrial membrane corresponding to hundreds of kilovolts per centimeter of the membrane thickness.
The science that this book describes still contains many blank spots. It is
unclear what the molecular mechanisms of the interconversion of the majority of
different energy forms are. Even more, the schemes in Fig. 1.6 do not describe all
the possible types of biological energy transformation. For instance, they lack the
generation of light. Light is not just an energy source. All living beings generate
photons that are produced in the course of free radical reactions that take place in
living cells. It is not clear whether this is just a byproduct of such reactions, or
whether cells use such light as a kind of signaling.
A very vivid illustration of the depth of our ignorance is the history of the discovery of the role of mitochondria in programmed cell deathapoptosis. At the very
end of the twentieth century, death proteins were discovered in the mitochondrial
intermembrane spacethey trigger apoptosis when leaving the mitochondria into
the cytosol. Hence mitochondria are not only the electric stations of cells, but also
destiny-defining organelles. And one of the simplest respiratory chain electron
carrierscytochrome c, which has been studied by so many biochemists starting
from David Keilin who discovered it in 1925proved to be a key player in the
cell suicide scenario. It is important to note that this property is a completely different
function of cytochrome c that has nothing to do with its role as an electron carrier
(see Chap. 15).
14
1 Introduction
15
two ADP molecules into one molecule of ATP and one of AMP. The standard
energy price of the pyrophosphate bond in the terminal phosphate of ADP is
similar to that in ATP. So it seems that it would have been possible to base all of
cell energetics on the hydrolysis of ADP to AMP. But for some reason evolution
chose the more complicated ATP, and not the simpler ADP. This paradox can be
explained if we look at two ways of ATP hydrolysis (Eqs. 1.5 and 1.6).
ATP H2 O ! ADP Pi
1:5
1:6
Terminal phosphate hydrolysis of ATP is shown to take place when the energyconsuming process driven by the ATP hydrolysis requires energy less than or
equal to 10 kcal mol-1. This is also the case when the energy requirement is much
larger than 10 kcal mol-1. In the latter case, a special mechanism (e.g., actomyosin filament) is at work making it possible to use the energy of many ATP
molecules simultaneously to perform a functional act.
If the energy requirement is only slightly higher than 10 kcal mol-1, i.e., by
several kcal mol-1, ATP is hydrolyzed to AMP and inorganic pyrophosphate (see
below, Sect. 7.3). In the living cell, the energy release is usually 45 kcal mol-1
higher when AMP and PPi are formed instead of ADP and Pi. This is because of a
much lower cytosolic concentration of PPi than of Pi. This effect is the result of
hydrolysis of PPi by soluble pyrophosphatases.
So, we can state that the use of adenosine triphosphate rather than adenosine
diphosphate (also a high-energy compound) as a convertible energy currency adds
some flexibility to the biological energy supply system (Skulachev 1988).
16
1 Introduction
absorb visible light) is very small, while ultraviolet light is absorbed by almost all
chemical compounds. Ultraviolet photons could initiate many chemical reactions
between simple substances that were present in the primordial ocean and ancient
Earths atmosphere. The American biochemist Cyril Ponnamperuma conducted
the following experiment. He put a sterile solution of hydrocyanic acid into an
ampule, sealed it, and then irradiated it with ultraviolet light. Adenine and other
purines and pyrimidines were formed in the solution as a result of the irradiation
(Ponnamperuma et al. 1963; Ponnamperuma 1966).
According to Ponnamperuma, ultraviolet also caused synthesis of adenosine
under the same conditions, and if ethyl metaphosphate was added to hydrocyanic
acid, then AMP, ADP, ATP, and adenosine tetraphosphate were synthesized. In
the latter case the ADP ? ATP transformation was much more effective than
AMP and adenosine synthesis or AMP ? ADP transformation.
When modeling the absorption of the ancient Earths atmosphere, Carl Sagan
came to the conclusion that it had a gap in the 240290 nm region and was thus
transparent for ultraviolet light. This can be explained by the fact that the main
simple compounds of that atmosphere (H2O, CH4, NH3, CO2, CO, and HCN)
absorb light of wavelengths shorter than 240 nm, and the absorption maximum of
formaldehyde (which is also supposed to have been part of that original atmosphere) occurs at wavelengths longer than 290 nm. It is to this gap that the
spectral maxima of purines and pyrimidines belong (Ponnamperuma et al. 1963;
Sagan 1966; Ponnamperuma 1966).
Here are the reasons why, according to Sagan and Ponnamperuma, that adenine
has certain advantages in serving as an ultraviolet light antenna when compared to
other purines and pyrimidines: (1) the strongest light absorption in the mentioned
spectral gap; (2) higher resistance to the destructive effect of ultraviolet light,
and (3) longer duration of excited state caused by absorption of an ultraviolet
quantum (Ponnamperuma et al. 1963; Sagan 1966).
Lev Blumenfeld and Mikhail Temkin (1962) calculated that the magnitude of free
energy changes accompanying the disruption of the aromatic structure of adenine are
similar to the energy required to form ATP from ADP and inorganic phosphate.
On the basis of all the above-mentioned data, we suggested the following
mechanism of ultraviolet light-based phosphorylation in primordial living cells
(Skulachev 1969, 1994, 1996).
(1) The adenine part of ATP absorbs an ultraviolet quantum, which leads to an
excited state with a disturbed system of double bonds. At the same time, the
amino group of adenine, which normally corresponds to an aromatic amine,
adopts the properties of an aliphatic group, which makes it easier for a
phosphorus atom of inorganic phosphate to attack it.2
There is also another possibilityphosphate being connected to N1 of adenine and not to its
amino group. For instance, natural compounds where NAD+ is connected to ribose in N1 position
have been described (Lee et al. 1989).
17
(2) Excited adenine of ADP is phosphorylated, thus producing a PADP, the ATP
isomer with the third phosphoryl group at the adenine amino group.
(3) A phosphoryl group is transferred from adenine to the terminal (second) ADP
phosphate. This transfer is enhanced by the fact that the distance between the
adenine and the second phosphate ADP is exactly equal to the size of another
(the third) phosphate residue (it was Szent-Gyrgyi who was the first to take
note of this fact (Szent-Gyorgyi 1957)). The transfer of the phosphoryl group
from the adenine head of the nucleotide to the phosphate tail is coupled
with its stabilization, because a very labile phosphoamide is being replaced
with a less labile phosphoanhydride (Fig. 1.7).
Stages (2) and (3) are hypothetical; they are supposed to explain the mechanism
of ultraviolet-induced ATP synthesis in Ponnamperumas experiments (Ponnamperuma et al. 1963; Ponnamperuma 1966).
Adenine and less often other purines and pyrimidines are known to be part of
key coenzyme and enzyme prosthetic groups, such as nicotinamide adenine
18
1 Introduction
19
has the structure of a bilayer of two phospholipid molecule leaflets that contact
each other with their hydrophobic (hydrocarbon) tails, while the hydrophilic
heads (phosphate residues) of the phospholipids are on the two opposite
membrane surfaces. The thickness of such membranes is about 50 (Skulachev
1988, 1994).
Formation of the first membranes meant the creation of the first cells, the inner
content of which was separated from the outer environment with a sufficiently
reliable barrier. Appearance of these tiny separate vesicles could have played an
important role in protection from unfavorable effects of ultraviolet radiation.
20
1 Introduction
21
22
1 Introduction
D
lH could reverse the H -ATPase reaction that had been used earlier for outward
transport of protons produced by glycolysis. In this way, the FoF1 complex might
have been transformed from ATPase to ATP-synthase (Fig. 1.10).
The structure of bacteriorhodopsin is much simpler than that of the system of
chlorophyll photosynthesis. The protein part of bacteriorhodopsin consists of one
23
24
1 Introduction
Fig. 1.11 Chlorophyll-based photosynthesis of purple bacteria (simplified scheme). Explanations are given in the text
25
scheme). Explanations are given in the text. H C OH represents a part of the glucose molecule
j
reduces intracellular carbon dioxide to glucose. Protons are taken from the cytosol
during this process. Extracellular water that is cleaved to O2 and H+ serves as a
reductant of the photooxidized chlorophyll. The inward H+ flow via FoF1 is coupled to generation of ATP (Fig. 1.12). So, cyanobacterial photosynthesis produces
not only ATP, but also carbohydrate, the main reserve substance of present-day
living cells. Cyanobacteria are the most probable evolutionary predecessors of
chloroplasts, the green plant organelles that function on the basis of the same
scheme shown in Fig. 1.12.
26
1 Introduction
(simplified scheme). Explanations are given in the text. H C OH represents a part of the glucose
j
molecule
The respiratory chain provides a mechanism for oxidation of respiratory substrates. These substrates can be obtained, for example, from glucose, which is in
turn produced by photosynthetic organisms. We can say that respiratory chain
enzymes fulfill functions that are opposite to those of cyanobacterial and chloroplast photosynthesis. It is not synthesis, but oxidation of organic substances that
occurs in the course of respiration. As a result, O2 is reduced to water instead of
H2O being oxidized to O2. However, in both cases ATP synthesis is coupled to
downhill H+ transfer (Fig. 1.13).
Like chloroplasts being developed from cyanobacteriaanimal, plant, and
fungal mitochondria probably originated from aerobic bacteria. That is why it is
not surprising that the functions of the mitochondrial respiratory chain can be
described by the scheme of bacterial respiration presented in Fig. 1.13.
Replacement of H+ by Na+ in a number of bioenergetic structures was probably
the final step in the creation of the energy scheme of modern living beings. Sodium
ions have two advantages when compared to protons. First, sodium is far more
widely available (compare 5 9 10-1 M Na+ to 1 9 10-7 M H+ in the oceans
water). Second, membranes are more permeable for H+ than for Na+. Proton
acceptor groups of membrane proteins increase the H+ permeability of membranes,
while this does not occur in the case of Na+. It is very difficult to create a
membrane which is not permeable for H+it is much easier to do this for Na+.
27
And protons are especially difficult to use as coupling ions under alkaline conditions when their concentration is very low. Protons are also inconvenient for life
at high temperatures, for this factor increases H+-permeability of membranes much
more than their Na+-permeability. If we apply a principle well-known to enzymologistsprotein has unlimited abilitiesthen we can imagine a protein that
facilitates outward transport of Na+ at the expense of some energy source as well
as another protein that allows Na+ to enter the cell, the latter process being coupled
to ATP synthesis or performance of some other useful work. In fact, such
sodium membranes use Na+ instead of H+ as the coupling ion. For instance,
there are membranes of some marine bacteria that have a Na+-transporting
respiratory chain (or Na+-decarboxylase) and Na+-ATP-synthase. There are also
external membranes of animal cells with Na+/K+-ATPase and Na+-dependent
proteins that function as carriers of different substances (see Chaps. 12 and 13).
Appearance of calcium energetics might have been one more step of evolution. Bivalent calcium ions fulfill two functions in the majority of organisms
structural (comprising skeletons) and regulatory (facilitating reversible change in
properties of a variety of extracellular and intracellular components). Different
systems of calcium ion transport (Ca2+-ATPases, Na+/Ca2+-exchangers, Ca2+channels) are present in the membranes of all the main organelles of cells (plasmalemma, sarcoplasmic reticulum, endoplasmic reticulum, Golgi apparatus, and
mitochondria). Part of the energy production in animal cells is always spent to
support Ca2+ flows that regulate such important processes as nerve impulse
transfer and intracellular hormone signaling, initiation of muscle contraction and
cell growth regulation, stimulation of anaerobic and aerobic catabolism, secretory
cell exocytosis and intermembrane contact formation, control of cell aging and
death, etc. One of this books authors (F.O.K.) obtained some data suggesting that
calcium ion transmembrane gradients can stabilize for some time the DW level on
cell membranes when D
lH -generators are inhibited (Kasparinsky and Vinogradov
1996). Current data on the spacial organization of close contacts of potentialdependent plasmalemma Ca2+ channels, endoplasmic reticulum, and mitochondria
suggest the hypothesis of direct delivery of calcium gradient free energy to many
cell compartments.
References
Belitser VN, Tsibakova ET (1939) On the coupling mechanism of the respiration and
phosphorilation. Biokhimiya (Russ) 4:516521
Blumenfeld LA, Temkin MI (1962) On possible mechanism of formation of adenosine
triphosphoric acid in oxidative phosphorylation. Biophysika (Russ) 7:731734
Engelhardt WA (1930) Ortho- und Pyrophosphat im aeroben und anaeroben Stoffwechesel der
Blutzellen. Biochem Z 251:1621
Engelhardt WA (1931) Anaerobic decomposition and aerobic resynthesis of pyrophospate in
erythrocytes of birds. Kazan Med Zhurn (Russ) 27:496504
Engelhardt WA (1932) Die Beziehungen zwischen Atmung und Pyrophosphatumsatz in
Vogelerythrocyten. Biochem Z 251:343345
28
1 Introduction
Part II
Chapter 2
Generators of D
lH play a leading role in the conversion of external energy
sources into forms that can be used by living cells. We begin by discussing lightdependent (photosynthetic) generators. First of all, we consider them to have been
the primordial systems for proton potential generation driven by external energy
sources during the evolution of life. Second, photosynthesis still plays the key role
in energy supply to the biosphere. The biosphere is known to consist mainly of
photosynthetic organisms and organotrophs that directly or indirectly consume
products of photosynthesisorganic substances and oxygen. Oxygenic photosynthesis, i.e. photosynthesis that produces oxygen, takes place in green plants and
cyanobacteria. It is this type of photosynthesis that provides oxygen for the planet.
One can point to the precise geographic coordinates of the areas of the Earth that
are responsible for this function. Most of the Earths oxygen is produced in the
taiga forests in Siberia. Canada, with its temperate zone forests, especially
coniferous ones, provides a smaller contribution. The famous tropical forests
(African and South American jungles) as well as the Worlds ocean do not play an
important role in this process due to the presence of vast numbers of heterotrophic
bacteria that absorb that very oxygen that is produced by photosynthetic organisms. So, Siberia can be compared to the planets lungs, and if Siberian forests are
destroyed it will mean the end of aerobic life on Earth.
There is one other technical but nevertheless important point that explains our
decision to start our presentation with photosynthesis. This process is initiated by a
quantum of light, and this fact makes it possible to use modern physical instrumentation for the analysis of its mechanism. For instance, when using an ultrafast
laser one can generate a 2 9 10-14 s long flash of light so as to study kinetics and
mechanisms of the primary processes of light energy storage.
31
32
33
to the main orbital. Such a return is accompanied with dissipation of the absorbed
photon energy in the form of heat or in the form of a light of longer wavelength
(fluorescence or phosphorescence). But photosynthesizing organisms have learned
to use the effect of chromophore transition into an excited state for the storage of
the energy of the quantum (hv) in a form which is useful for a cell. For instance, if
during the excited state lifetime an electron is transferred from the excited orbital
to some acceptor X, and the vacancy on the main chromophore orbital is filled
from some donor Y, then part of the energy of the photon can be stored as a
difference of redox potentials of substances X and Y under the condition that the
redox potential of the Yoxidized/Yreduced couple is more positive than that of the
Xoxidized/Xreduced couple. Even more so, if this redox reaction is organized in such a
way that an electron is transferred from Y to X across the membrane, then a
certain part of the energy of the light quantum can also be stored as D
lH . Just in
this way the most important photosynthetic process, i.e. oxygenic photosynthesis,
is organized.
In an alternative and simplified version of photosynthesis, an electron from an
excited orbital is returned to the main orbital, but not directly. It has to cross the
membrane first, which leads to generation of D
lH . This type of photosynthesis is
found in purple bacteria.
34
Fig. 2.3 a, b Structure of the reaction center complex (RC) and of antenna I (LH-I) of purple
bacteria. Bacteriochlorophylls are represented as rhombs. Bacteriochlorophylls of antenna I are
shown in green color; (BChl)2 (it is labeled PA); and bacteriochlorophyll monomers (BA and BB)
of the reaction center are red and blue, respectively. The a-helical columns of the reaction center
subunits are presented as colored rodsyellow (L-subunit), red (M-subunit), and gray
(H-subunit). Antenna I proteins are shown as blue and pink rods. Bacteriopheophytins are not
shown. c Structural arrangement of the reaction center complex, antenna I, and antenna II in
purple bacteria. The polypeptide chain of the proteins and bacteriochlorophyll molecules are
shown. (Hu et al. 1998)
excitation energy to the antenna bacteriochlorophyll. One should note that in the
case of acidophilic bacteria Mg2+ ions are replaced by Zn2+ ions in bacteriochlorophyll, so that the Mg2+ of the porphyrin complex would not be replaced by
H+ ions, which would otherwise occur under acidic conditions (Wakao et al.
1996). The excitation migrates through antenna bacteriochlorophyll until it reaches
what is called a special pair, a bacteriochlorophyll dimer bound to other proteins forming a photosynthetic reaction center complex (Fig. 2.3).
Besides the dimer, the complex contains two bacteriochlorophyll monomers,
two bacteriopheophytins (i.e. bacteriochlorophyll analogs where the Mg2+ ion is
replaced by two H+ ions), and two ubiquinone molecules (CoQ).1 The amount of
reaction center pigment complex is usually about two orders of magnitude smaller
than that of the antenna pigment complex.
The excited bacteriochlorophyll dimer serves as an electron donor to ubiquinone (CoQ). This process is directed across the membrane to its cytoplasmic side.
It includes a number of intermediate stages with consecutive reduction and oxidation of bacteriochlorophyll monomer, bacteriopheophytin, and bound MQ and/
or CoQ (reactions 14 in the scheme of the cyclic redox chain, Fig. 2.4).
In certain bacteria, one of the ubiquinone molecules is replaced by a menaquinone (MQ); see
structure in Appendix 2.
35
For reduction, CoQ needs two electrons and two protons (Fig. 2.5). One of
these electrons is donated by the reaction center and the other by heme bH of
cytochrome b.2 Having accepted two electrons, CoQ combines with two protons
from the nearest water phase (above the membrane, i.e. in the cytosol in Fig. 2.4;
CoQH2 formation is described by reaction 5 in Fig. 2.4 and in more detail in
Fig. 2.5).
When formed, CoQH2 diffuses across the membrane in the direction of its
opposite (periplasmic) side (Fig. 2.4, reaction 6). Close to this membrane surface,
CoQH2 is oxidized to the semiquinone form (reaction7) by the nonheme iron
sulfur (FeS) protein abbreviated as FeSIII. When CoQH2 is oxidized, two H+ ions
are released to the periplasm or inside the chromatophore (Fig. 2.4).
The electron accepted by FeSIII is later used for the reduction of the bacteriochlorophyll dimer that was previously oxidized during the first stage of the
cycle. Cytochromes of c type participate in the electron transfer from FeSIII to
(BChl)+
2 (reactions 810; in the case of Rhodospirillum rubrum these are cytochromes c1 and c2).
The radical ion CoQ that appears after CoQH2 has lost one electron and two
protons reduces heme bL (reaction 11). The latter transfers the electron to the
Cytochromes are heme-containing proteins catalyzing redox processes (for heme structures,
see Appendix 2). Cytochromes having the same heme group and differing in the protein moiety
are usually indicated by the same letter but different numerical indexes.
36
37
Fig. 2.6 Redox potentials of electron carrier involved in the photosynthetic systems of purple
bacteria (a) and green sulfur bacteria (b). For explanations, see Sects. 2.1.1 and 2.2.2, respectively
258, 323, and 273 amino acid residues, respectively), which is in obvious contrast
to the previously accepted nomenclature. The old subunits names are nevertheless
still used in the literature.
The hydropathy profile of the M-subunit shows five very hydrophobic columns
composed of at least 20 amino acid residues each. Each column is long enough to
span the membrane in an a-helix. This conclusion was directly confirmed by X-ray
analysis of Blastochloris viridis reaction center complexes (Hartmut Michel,
Johann Deisenhofer, and Robert Huber, Nobel Prize in Chemistry, 1988, Fig. 2.7).
They obtained 0.51-mm crystals of the complexes using (NH4)2SO4 treatment of
the reaction center complex solution supplemented with a detergent.
An X-ray study of these crystals revealed for the first time the threedimensional structure of a membrane energy transducer at 0.3-nm resolution
(Deisenhofer et al. 1984, 1985a, b). It was found that the 13-nm-long complex is
composed of two peripheral domains (tetraheme cytochrome c and the H-subunit)
and the central part (M- and L-subunits). It is the M- and L-subunits that bear
bacteriochlorophylls and bacteriopheophytins of b-type, quinones, and nonheme
iron that is connected to imidazoles of the four histidines of the protein. Comparing these data with earlier observations, one can assume that cytochrome
c faces the periplasm, the H-subunit the cytoplasm, the M- and L-subunits being
integrated in the hydrophobic membrane layer.
The X-ray picture of the reaction center complex reveals eleven transmembrane
a-helical columns that are oriented perpendicular to the plane of the membrane and
parallel to the long axis of the complex, being located in its middle (membrane)
part. The H-subunit has only one a-helical column, while the M- and L-subunits,
being similar to each other in their amino acid sequences and spatial structures,
contain five such columns each. These columns are oriented symmetrically about
the longitudinal axis of the complex (Fig. 2.8).
The single hydrophobic a-helix of the H-subunit serves as an anchor binding
this hydrophilic polypeptide to the membrane. Another peripheral subunit, tetraheme cytochrome c, it is attached to the membrane by contacts with other subunits
38
Fig. 2.7 Nobel Laureates Hartmut Michel, Johann Deisenhofer, and Robert Huber
and by two fatty acids covalently bound to a glycerol residue forming a thioether
bond with the SH group of the N-terminal cysteine of this cytochrome (Fig. 2.9).
When looking at the packing of polypeptide chains of the H-, M-, and
L-subunits in the membrane, one should note complete absence of charged amino
acids residues (Fig. 2.10a) and almost complete absence of bound water
(Fig. 2.10b) in the transmembrane a-helical columns. Such amino acids and water
are located on the membrane surfaces; the majority of the negatively charged
residues are found on the outer membrane surface, whereas positively charged
amino acids are exposed on the inner membrane surface. This fact suggests that the
assembly of the reaction center complex polypeptides inside the membrane is
directed by transmembrane DW (the cell interior is negative). Electrophoresis of
the protein domains is apparently involved in this assembly.
It should be noted that crystallization of the membrane proteins is extremely
difficult to perform. The success in attempts to crystallize the Bl. viridis reaction
center complex seems to be due to the fact that its central hydrophobic part (the
M- and L-subunits) is to some extent protected from water by large hydrophilic
polypeptidesthe H-subunit and tetraheme cytochrome c.
Arrangement of Redox Groups. The most important result of the X-ray study of
the Bl. viridis reaction center complex is that the arrangement of redox prosthetic
groups is revealed (Deisenhofer et al. 1984). In Fig. 2.11, the results of the X-ray
analysis of the complex is shown in such a way that only prosthetic groups are
seen. According to these data, four cytochrome c hemes are located above
the bacteriochlorophyll dimer. The FeFe distances between the hemes are
1.41.6 nm. The Fe atom of the heme closest to (BChl)2 is situated 2.1 nm above
two Mg atoms of the bacteriochlorophyll dimer. The dimer is oriented parallel to
the long axis of the reaction center complex. It is fixed between two a-helical
D segments belonging to the M- and L-subunits (DM and DL, respectively). In each
a-helical column there is a histidine residue, the imidazole group of which serves
as an axial ligand for Mg2+ in (BChl)2.
Two bacteriochlorophyll monomers were found to be arranged slightly below
the dimer (the distance between Mg2+ ions in (BChl)2 and BChl is 1.3 nm, and the
39
Fig. 2.8 Structure of protein subunits of the Blastochloris viridis reaction center: I, II, III, and IV
stand for tetraheme cytochrome c, L-, M-, and H-subunits, respectively; Vgeneral view of the
reaction center complex; yellow electron-transporting prosthetic groups; VIcross-section of the
complex showing relationship between 11 a-helical segments (top view; A, B, C, D, E, five
transmembrane a-helical segments of M- and L- subunits). The region occupied by the
chromophores is shaded. The segments are tilted by an angle of 38 (segments D) and \ 25
(other segments) to the membrane plane. Figure section VI shows a section at a level close to the
center of the hydrophobic membrane layer (Deisenhofer et al. 1984; Henderson 1985)
angles between the porphyrin ring planes are * 70). Imidazoles of histidine
residues located in CM and DM or CL and DL segment connections are ligands for
Mg2+ of the monomers. Bacteriochlorophyll monomers are in contact with
40
41
Fig. 2.10 Structure of reaction center complex of Blastochloris viridis. acharged amino acid
residues in the complex. Positively charged amino acids are orange, and negatively charged ones
are blue. bbound water molecules in the reaction center complex of Bl. viridis. The central part
of the complex, which is immersed in the membrane, contains neither charged amino acid
residues nor water, this fact being responsible for the sharp decrease in dielectric constant and,
hence, small dissipation of energy occurring during the transmembrane electron transfer in this
part of the complex (Deisenhofer et al. 1984)
Connections between segments AM and BM, CM and DM, AL and BL, and CL and
DL as well as four of the five initial amino acid residues from the N-terminus of the
H-subunit polypeptide are in contact with tetraheme cytochrome c. The Tyr-L162
residue is located between (BChl)2 and the nearest heme. It is supposed to participate in the electron transfer from this heme to the oxidized (BChl)2. The GluH177 residue, which has access to the CoQ pocket, is presumably involved in H+
transfer from the cytoplasm to CoQ-. Removal of the H-subunit was shown to
exert a strong effect on the functioning of the reaction center at the level of CoQ.
According to spectral studies, the bacteriochlorophyll dimer is arranged perpendicularly to the plane of the Bl. viridis cytoplasmic membrane. Therefore, it
seems highly probable that the long axis of the reaction center complex is arranged
across the membrane. The distance between the Mg atoms of the bacteriochlorophyll dimer and the menaquinone is about 3.0 nm, so that an electron, running
from the dimer to the quinone, crosses a major part of the hydrophobic barrier of
the membrane.
42
Fig. 2.11 Location of prosthetic groups in the reaction center complex of Bl. viridis (X-ray
analysis data). Four hemes (above the membrane), bacteriochlorophyll dimer, two bacteriochlorophyll monomers, two bacteriopheophytins, menaquinone (MQ), and nonheme iron (Fe) are
shown (Deisenhofer et al. 1984)
43
that excitation of (BChl)2 is transferred when (BChl)2 is not oxidized by the right
branch and is transformed instead from a singlet to the more stable triplet state.
The excitation of the carotenoid is in turn dissipated through heat or emission of a
light quantum of a longer wavelength (fluorescence). If oxygen manages to attack
and oxidize the excited carotenoid, it leaves the reaction center complex so as to be
exchanged for a new (intact) carotenoid molecule. In such way the carotenoid
molecule sacrifices itself to save (BChl)2, BChl, or BPheo, the oxidation of
which would lead to irreversible inactivation of the whole complex.
To conclude this section, we note that the X-ray analysis data confirmed the
chromophore arrangement scheme (Fig. 2.12) postulated by Shuvalov and Asadov
in 1979 on the basis of optical spectroscopy (Shuvalov and Asadov 1979).
Sequence of Electron Transfer Reactions. The X-ray study shows the electron
pathway through the reaction center complex to be determined by the spatial
arrangement of redox groups:
hv
2:1
44
Fig. 2.13 Fast kinetics of light absorption changes (DA) in modified Rhodobacter sphaeroides
reaction center complexes lacking one of two bacteriochlorophyll monomers (BChlM).
Absorption decrease at 930 nm (A930) indicates formation of the excited bacteriochlorophyll
dimer (BChl)*2; A875 decrease indicates formation of (BChl)+2 . A805 decrease, reduction of
bacteriochlorophyll monomer, (BChl)L. A755 decrease, reduction of bacteriopheophytin, BPheo.
Initial A775 increase is a result of the BChl2 ! BChl2 transition. Excitation was induced by a
laser flash (t1/2 = 0.2 ps). (Matveetz et al. 1986)
difference. Its potential is about 950 mV, i.e. even slightly more negative than in
the case of the nonexcited monomer, and even more so when compared to nonexcited bacteriopheophytin (about 750 mV).
As shown in the group of one of this books authors (V.P.S.), the heme of the
tetraheme cytochrome c, serving as a (BChl)+
2 reductant, is characterized by an
a-band at 559 nm and a redox potential of about +380 mV (Dracheva et al. 1986).
One can speculate that this heme is located just above (BChl)2 in the reaction
center complex. Heme559 is, in turn, reduced by heme556 (whose redox potential is
about 310 mV). The electron donor for heme556 is most probably water-soluble
cytochrome c2. The role of the two other hemes of the tetraheme cytochrome
c (redox potentials +30 mV and 50 mV) remains obscure.
The most remarkable feature of the primary processes of photosynthetic electron transfer is their extremely fast rates. These processes represent the fastest
chemical reactions known in biological systems thus far. As a result, the first
intermediates of the photoredox chain are very short-lived, which is needed to
prevent their oxidation by oxygen.
In Bl. viridis, anion-radical MQ- is the first intermediate that has a turnover rate
comparable to that of the intermediates of the majority of enzymatic processes.
Oxidation of MQ- by bound CoQ takes about 0.1 ms. Therefore, MQ is often
45
described as a primary electron acceptor. In some bacteria, this role is played not by
MQ, but also by CoQ. In these cases the reaction center complex contains two
molecules of CoQthe primary acceptor CoQA and the secondary acceptor CoQB.
Electron transfer between the two quinones is facilitated by the presence of a
nonheme iron that does not undergo oxidoreduction and persists in the Fe2+ form.
In fact, Mn2+ can effectively substitute for Fe2+ in the reaction center complex
(Okamura et al. 1982).
From one point of view, when looking at the mechanism of electron transfer in
the reaction center complex, CoQB can be considered the final product of the
operation of the complex:
Q
A QB ! QA QB
2:2
More probable, however, is that not only QB, but also QB is reduced by QA
(see Eq. 2.3). Supporting this version, one should note that the affinity of QB for
the L-subunit of the photosynthetic reaction center complex is much higher than
that of QB and QBH2.
Reduction of QB is accompanied by addition of two protons:
Q
A QB 2 H ! QA QB H2
2:3
Mechanism of D
lH Generation. Because cytochrome c2 and quinone are
located on the opposite sides of the membrane (Okamura et al. 1982; Deisenhofer
et al. 1984), the transfer of reducing equivalents is directed through the membrane
to its cytoplasmic surface. Assuming that it is an electron that moves from cytochrome c2 to quinone, one can predict that the cytoplasm should be charged
negatively relative to the periplasm or the interior of the chromatophore.
This prediction was confirmed by a series of observations concerning DW
generation by the reaction center complexes. To monitor DW, L. Drachev, A.
Kaulen, and A. Semenov in our group developed a method that made it possible to
carry out direct voltmeter measurements of DW generated by enzymes built into
proteoliposomes (Drachev et al. 1974, 1979). The term proteoliposome was
suggested by one of the authors of this book (V.P.S.) in 1972 for closed membrane
vesicles that could be obtained as a result of self-assembly from phospholipids and
proteins (Skulachev 1972; Kayushin and Skulachev 1974). In these experiments,
proteoliposomes were sorbed on one of the surfaces of a collodion film that was
soaked with a solution of phospholipids in decane.
The experiments showed that proteoliposomes are sorbed in such a way that
their inner solution does not mix with the outer one (Severina 1982). It is likely
that during sorption a part of the proteoliposome membrane that is in contact with
the film is dissolved by the decane the film is soaked with, while other parts of the
membrane remain intact, thus forming a barrier between the inner solution of the
film-attached proteoliposome and the outer solution surrounding the film.
The generation of DW on the membrane of sorbed proteoliposomes can be
registered by a voltmeter by using two electrodes that are placed on the two sides
of the collodion film. The time resolution of this system is about 50 ns, which is
46
47
48
Fig. 2.15 Generation of DW by Bl. viridis reaction center complexes in response to two
consecutive 15-ns laser flashes. The time between flashes was 0.5 s. Only the second flash
generates a slow electrogenic phase developing on the microsecond time scale (a). This phase
is shown at higher resolution in (b), where dashed and solid lines represent the first and second
flashes, respectively. DW was measured with the proteoliposome-collodion film system. CoQ10
was added to the solution used to impregnate the film. The redox potential of the medium was
+240 mV. (Dracheva et al. 1986)
or H+ ions are transferred through a less hydrophobic part of the complex. Most
likely both factors contribute to this phenomenon. It seems quite probable that a
similar situation occurs during the stages of electron transfer.
The distances between QA and (BChl)2, (BChl)2 and c559, and c559 and c556
along the axis vertical to the membrane plane are 2.9, 2.1, and 1.2 nm, respectively. If the electrogenesis were a linear function of the distance between the
redox groups involved, the relative contributions of these electron transfer steps
would be 1:0.7:0.4. In fact, they were found to be 1:0.2:0.7. This means that the
contribution is greater when the electron transfer stage is immersed deeper into the
membrane. This pattern becomes quite understandable when we take into account
the fact that the membrane dielectric constant value increases approaching the
water phase (Skulachev 1988).
In the 1960s, when Peter Mitchell formulated his chemiosmotic hypothesis, he
suggested that an electron moving from cytochrome c to CoQ via bacteriochlorophyll crosses the hydrophobic membrane barrier (the electron transfer half-loop)
(Mitchell 1966). This assumption, quite speculative at that time, has now been
directly proved. The only new point that was detected in the result of experimental
trial of the original Mitchell scheme is that besides the electron transfer across the
membrane, there is a small but measurable electrogenic phase arising because of
the movement of the proton in the opposite direction (this movement being necessary for formation of CoQH2 from CoQ) (Skulachev 1988).
49
Fig. 2.16 Generation of DW by Bl. viridis reaction center complexesthe role of high-potential
hemes of cytochrome c. Measurements were carried out at redox potential of the medium equal to
+220 mV (two high-potential hemes of the tetraheme cytochrome c were reduced and two lowpotential hemes were oxidized), +380 mV (one of high-potential hemes was 50 % reduced), and
+440 mV (all four hemes were oxidized). The figure to the right (b) shows part of figure (a) in
more detail. (Dracheva et al. 1986)
50
Fig. 2.17 Mechanism of electrogenic phases in Bl. viridis reaction center complex (Dracheva
et al. 1986; Skulachev 1988)
By analogy with the more elaborate mitochondrial complex, one can suppose
that D
lH generation by this system is partially due to the electron transfer from
heme bL to bH, directed perpendicularly to the membrane plane, as shown in
Fig. 2.4. This process is oriented in the direction of the cytoplasmic surface of a
bacterial membrane. An electron was shown to cross 3540 % of the membrane
thickness while moving from heme bL to bH (Glaser and Crofts 1984). The rest is
assumed to be crossed by H+ ions moving in the direction opposite to that of an
electron (see Fig. 2.4).
It remains unclear whether CoQ, CoQ-, or both serve as electron acceptor(s)
for cytochrome bH. If this function is specific only for one of these components, it
means that formation of a completely reduced form of ubihydroquinone (CoQH2)
must involve the dismutation reaction (2 CoQ- ? 2 H+ $ CoQ ? CoQH2). The
reaction center complex is another source of electrons for CoQ reduction to
CoQH2. Steady-state functioning of the system requires that for each two CoQ
51
molecules reduced to CoQH2, one would be reduced by the reaction center and the
other by bH heme. Protons required to convert CoQ to CoQH2 are in both cases
transported from the cytoplasm.
Having been formed, CoQH2 crosses the greater part of the hydrophobic
membrane barrier to be oxidized by FeSIII. As a result, CoQ-, reduced FeSIII, and
2 H+ are formed, the latter being transported through the remainder of the
hydrophobic barrier to be released to the periplasm or chromatophore interior. In
the case of Rh. rubrum and Rhodobacter sphaeroides, electron transfer from FeSIII
to (BChl)2 via cytochrome c1 and cytochrome c2 completes the cycle, as shown in
Fig. 2.4. In Bl. viridis, tetraheme cytochrome c participates in the electron transfer
between cytochrome c2 and (BChl)+
2 .
The reaction sequence catalyzed by the CoQH2:cytochrome c-oxidoreductase,
as shown in Fig. 2.4, represents, in fact, a version of the Q-cycle scheme postulated by Mitchell (Mitchell 1975). The key postulate of this scheme was that
CoQH2 oxidation and CoQ reduction occur in such a manner that the fate of each
of the two electrons removed from CoQH2 or added to CoQ appears to be different.
Now this suggestion is firmly established at least for CoQH2 oxidation. FeSIII was
shown to act as a CoQH2 oxidase, whereas heme bL of cytochrome b was shown to
serve as an oxidase of CoQ-, the latter being formed from CoQH2. There is a
chemical reason for the fact that CoQH2 and CoQ- are oxidized by different
enzymesthe redox potentials of hydroquinone and semiquinone are quite different (Rich 1984).
It is noteworthy that the binding of CoQH2 to the FeSIII protein proceeds in
such a way that the hydroquinone head of CoQH2 is bound near a positively
charged group (presumably Fe3+). This induces an acidic shift of the pK value of
CoQH2, which makes its deprotonation easier. Having lost a proton, CoQH2
changes into CoQH-, which is oxidized by FeSIII. Deprotonation seems to be
necessary for this oxidation reaction since the redox potential of the CoQH2/
CoQH+
2 couple is higher than +850 mV, while that of CoQH /CoQH couple is
+190 mV (Rich 1984), which is more negative than that of FeSIII.
In mitochondria, electron transfer from ubiquinol to FeSIII and then via cytochrome c1 to cytochrome c is catalyzed by respiratory chain complex III, which is
also known as bc1 complex or ubiquinol:cytochrome c-oxidoreductase. This
complex has been found in a number of bacteria; in many respects it is similar to
plastoquinol:plastocyanin-oxidoreductase of thylakoids (bf complex). Redox
groups of the bc1 complex include an [2Fe - 2S] cluster, which is part of a socalled Rieske protein, two hemes of a b-type cytochrome and a cytochrome c1
heme. The [2Fe2S] cluster of the Rieske protein is connected to the polypeptide
by the bonds of one iron atom to two cysteine residues and of the other iron atom
to two histidine residues. Thus, the FeS cluster is part of the inner globular
structure of the polypeptide chain that is anchored in the membrane with its
hydrophobic N-terminal helix and is protruded from the lipid bilayer into the water
phase.
52
53
54
Fig. 2.19 Noncyclic photosynthetic redox chain of green sulfur bacteria (Hauska et al. 2001).
Explanations are provided in the text
2:4
while the process occurring on the inner membrane surface can be described as:
NAD + H 2e ! NADH:
2:5
55
56
iron connected to four imidazoles of histidines residues of the protein. The chlorophyll cation radical (ChlII)+
2 accepts an electron from one of the tyrosine residues of the protein, which, in turn, is reduced by an electron removed from a H2O
molecule by means of a rather complicated manganese-containing water-oxidizing
complex (WOC). Electrons, molecular oxygen, and protons are released when the
water molecule is oxidized. The released protons appear in the intrathylakoid
space. The reduced PQ is oxidized by the PQH2:plastocyanin-oxidoreductase (also
called b6f complex, which is a chloroplast analog of the bacterial and mitochondrial bc1 complex). The b6f complex comprises a three-heme cytochrome b (hemes
bL, bH, and ci), FeSIII, and cytochrome f, which is functionally similar to cytochrome c1.
The b6f complex catalyzes a Q-cycle similar to that described in purple bacteria
and mitochondria. As a result, the transport of one electron from PQ to cytochrome
f is coupled to the translocation of one charge across the thylakoid membrane, the
absorption of two H+ ions from the chloroplast stroma, and the release of two H+
ions into the thylakoid interior. These protons are taken up on the outer surface of
the thylakoid membrane and are released on its inner surface.
From cytochrome f the electron moves to the copper-containing protein plastocyanin (Pc), which reduces the cation radical of the chlorophyll dimer of photosystem 1 (in the ground state, the absorption maximum for this chlorophyll is
near 700 nm). The (ChlI)+
2 radical cation is formed as a result of the oxidation of
the excited (ChlI)*2. The electron taken from (ChlI)*2 is transferred via the
chlorophyll monomers (ChlI and ChlI0 ) and vitamin K1 (phylloquinone) to the
ironsulfur center FeSX,, which acts as the primary stable electron acceptor in
photosystem 1.
During the next stages the electron is transported to other ironsulfur clusters
(FeSB, FeSA, and ferredoxin) and further to a flavoprotein containing FAD as the
57
Fig. 2.21 Noncyclic light-dependent redox chain of chloroplasts (Blankenship and Prince 1985):
WOCwater-oxidizing complex; yellow, electron-transporting prosthetic groups; Ztyrosine
residue in a protein of photosystem 2; (ChlII)2dimer of photosystem 2 chlorophyll; ChlII
monomer of photosystem 2 chlorophyll; PQA and PQBplastoquinones bound to photosystem 2;
PQfree plastoquinone; bH and bLhigh- and low-potential hemes of cytochrome b6;
fcytochrome f; PCplastocyanin; (ChlI)2dimer of photosystem 1 chlorophyll; ChlI and
ChlII- monomers of photosystem 1 chlorophylls; K1vitamin K1; FeSXprimary stable electron
acceptor of photosystem 1; FeSA and FeSBtwo ironsulfur clusters tightly bound to
photosystem 1; Fdferredoxin. a Diagram illustrating the most probable arrangement of redox
centers in the thylakoid membrane. b Reducing equivalent carriers are arranged according to their
redox potentials; the dotted line shows the electron transfer pathway from ferredoxin to
b6f complex that provides cyclic electron transfer in the photoredox chain
prosthetic group. From FAD, the reducing equivalents come to NADP+. The
formed NADPH is oxidized by a water-soluble enzyme system reducing CO2 to
carbohydrates.
58
Within the framework of the scheme in Fig. 2.21, the noncyclic redox chain of
chloroplasts and cyanobacteria carries out transmembrane movement of 6 H+ per 4
quanta. This corresponds to the H+/e ratio of 1.5 (the ratio of the number of H+
ions transported across the membrane to the number of electrons transferred from
H2O to CO2). Thus, there are three D
lH generators in the thylakoid redox
chainphotosystem 1, photosystem 2, and the b6f complex.
2.3.2 Photosystem 1
Chloroplast photosystem 1 contains 14 different polypeptides. Two of them with
molecular masses of 83.2 and 82.5 kDa show 45 % similarity (percentage of
identical amino acid residues in the sequences). Other subunits are of substantially
lower mass (for review see (Okamura et al. 1982; Crofts and Wraight 1983; Nelson
and Ben-Shem 2004; Nelson 2011)).
(ChlI)2, ChlI, ChlI0 , vitamin K1 (phylloquinone), and FeSX are connected to
large subunits, while FeSA and FeSB are connected to one of the small hydrophobic subunits. In contrast to photosystem 1 of cyanobacteria, four proteins
carrying antenna chlorophylls (Lhca1-4) are integrated into chloroplast photosystem 1. When looking at the thylakoid membrane from the normal to the
membrane plane, one will note that antenna proteins form a half-circle around the
other subunits that constitute the reaction center complex of photosystem 1
(Fig. 2.22). The transfer of excitation energy from the antenna chlorophyll to
(ChlI)2 is extremely effectivealmost every photon absorbed by the antenna leads
to oxidation of (ChlI)2. The copper-containing protein plastocyanin (see below),
being part of photosystem 1, is located on the inner side of the thylakoid membrane, being absorbed by membrane surface. Another surface protein, ferredoxin
(11 kDa), is located on its outer side. This protein contains a FeS cluster and
transfers electrons from photosystem 1 to the flavoprotein that reduces NADP+
(34 kDa).
The photosystem 1 reaction center complexes form trimers in the thylakoid
membranes of cyanobacteria. In the case of higher plant chloroplasts, photosystem
1 is in the form of monomers. Both the trimers of cyanobacteria and the monomers
of chloroplasts have been purified, crystallized, and studied by X-ray structural
analysis. The results of X-ray studies of chloroplasts are shown in Figs. 2.22 and
2.23. Figure 2.23 shows two pathways of electron transfer through (ChlI)*2 to FeSX,
each containing the same redox groups (ChlI, ChlI0 , and vitamin K1). Both pathways are capable of electron transfer, but one of them is ten times faster than the
other.
The primary photoprocess in photosystem 1 is the electron shift from (ChlI)*2
(redox potential -1.2 V) to ChlI (redox potential about -1 V). The electron is
then transferred to ChlI0 and later to vitamin K1 (redox potential -0.8 V). FeSX
(-0.7 V) is the next electron carrier. It is a cluster of four iron ions and four sulfide
ions. In addition to this component, there are two more [4Fe - 4S] clusters (FeSA
59
and FeSB) in photosystem 1. Their redox potentials are -0.58 and -0.53 V,
respectively. The electron transfer sequence in this segment of the redox chain is
FeSX ? FeSA ? FeSB. The latter component serves as the reductant of the watersoluble protein ferredoxin that contains a [2Fe - 2S] cluster.
Another water-soluble electron carrier is the reductant of photooxidized
(ChlI)+
2 . This is plastocyanin, a copper-containing protein. The primary structure
of plastocyanin has sequences homologous to those in subunit II of cytochrome
oxidase, the terminal enzyme of the respiratory chain (see Sect. 4.4).
Plastocyanin is reduced by cytochrome f, which is a component of the
b6f complex. The latter transfers electrons from photosystem 2 to photosystem 1
(see below, Sect. 2.3.3).
There is an alternative function of the photosynthetic redox chain in chloroplasts,
namely cyclic electron transport, with photosystem 1 and b6f complex but not
photosystem 2 being involved. This system can be demonstrated in vitro when
photosystem 2 is blocked by the herbicide dichlorophenyl dimethylthiourea
60
(DCMU, also called Diuron) and an artificial electron carrier is added. Under certain
natural conditions, the cyclic electron transfer seems to be operative in vivo. It has
been proposed that in such case reducing equivalents are transported from ferre+
doxin back to (ChlI)+
2 instead of being accepted by NADP . This process involves
plastoquinone and the b6f complex. It is suggested that ferredoxin reduces PQ,
so photosystem 2 does not appear to be necessary to regenerate (ChlI)2 from
(ChlI)+
2 . This mechanism is similar to the cyclic redox chain of purple bacteria
generating D
lH without photolysis of water and reduction of NADP+ (Crofts and
Wraight 1983).
Photosystem 1 is clearly competent in generating D
lH . The reduction of
Photosystem 1 by plastocyanin was shown to occur on the inner side of the
thylakoid membrane, while ferredoxin and ferredoxin-NADP+ reductase, which
oxidize photosystem 1, are located on its outer side. Thus, the electron transport
via photosystem 1 must be directed across the membrane. The photosystem 1
complex has been reconstituted into proteoliposomes, which were then found to
generate D
lH under continuous illumination. To mediate cyclic electron transfer
around photosystem 1, the electron donor ascorbate and an artificial electron
carrier phenazine methosulfate were added to the incubation mixture. Incorporation of a purified H+-ATP synthase complex into the same proteoliposomes made
photophosphorylation possible (Nelson and Hauska 1979; Hauska et al. 1980).
61
The mechanism of D
lH generation mediated by photosystem 1 seems to be
similar to that mediated by bacterial reaction centers. A nanosecond laser flash
induces very fast (s \ 100 ns) DW generation, this process being much faster than
any reaction in photosystem 1 other than electron transfer from (ChlI)* to FeSX.
The generated DW composes a major part of the total electrogenesis linked with
the operation of photosystem 1, so all the other stages of electron transfer make
just a relatively small contribution to the energy transformation in this segment of
the photosynthetic redox chain (Witt 1979; Lopez and Tien 1984).
2.3.3 Photosystem 2
Photosystem 2 was found to consist (depending on the object and purification
method used) of about 30 different types of polypeptides, some of which are coded
by nuclear and some by chloroplast DNA. It forms dimers. The X-ray analysisbased structure of photosystem 2 and the location of redox centers are shown in
Fig. 2.24.
The photosystem 2 chlorophylls (similar to those from photosystem 1) belong to
the group of a-type chlorophylls and have an a-absorption band at 680 nm
(700 nm in case of photosystem 1). Subunits D1 and D2 (39.5 and 39 kDa,
respectively) construct the central part of photosystem 2. These subunits resemble
the M- and L-subunits of the purple bacteria reaction center complex (Hearst and
Sauer 1984; Cramer et al. 1985). They bind (ChlII)2, two ChlII, two pheophytins,
primary and secondary quinones (PQA and PQB), nonheme iron connected to four
histidines, and also two antenna chlorophylls (ChlZ). The 39-kDa subunit specifically binds DCMU.
The presence of 12 very small subunits (35 kDa) is characteristic of photosystem 2. Subunits of 9.5 and 4.5 kDa form the b-type cytochrome with a-band
maximum at 559 nm (cytochrome b559). Each of the mentioned subunits contains a
histidine residue with imidazole forming a coordinating bond with heme. The role
of cytochrome b559 remains unclear. The same can be said about the c-type
cytochrome (c550), which is located on the inner (directed toward the intrathylakoid space of the thylakoid) side of the membrane. This cytochrome is
present in cyanobacteria, but it is absent from chloroplasts. Cytochrome b559 is
located closer to the opposite membrane side.
A very high redox potential of the basic state of the chlorophyll dimer (the
(ChlII)2/(ChlII)+
2 couple) is a distinctive feature of photosystem 2 (Nelson and
Ben-Shem 2004; Nelson 2011). It is equal to +1.1 V. This explains why (ChlII)+
2
can serve as the electron acceptor for water (the redox potential of the H2O/O2
couple at neutral pH is ? 0.82 V). On the other hand, the redox potential of the
excited (ChlII)*2 is in negative (less than -0.6 V).
Plastoquinone (PQA) was shown to be the primary stable electron acceptor.
Another bound quinone molecule (PQB) participates in the further electron transfer
along the photosynthetic redox chain. Similar to the purple bacteria, this process is
62
Fig. 2.24 Chloroplast photosystem 2 structure. a Side view of photosystem 2 dimer: subunits D1
and D2 are in dark blue color, chlorophyll-binding antenna proteins CP43 and CP47 are red,
cytochrome b559 is light-blue, peripheral subunits orange and yellow, and other subunits pink.
b spatial arrangement of photosystem 2 prosthetic groups: chlorophylls are dark green,
manganese cluster light-blue, a water molecule bound to the manganese cluster pink, heme and
nonheme iron red, and quinones purple. The electron transfer pathway is indicated by arrows.
(Nelson and Ben-Shem 2004)
63
The mechanism of D
lH generation by photosystem 2 can be pictured by
analogy with the reaction center complex of purple bacteria that has already been
discussed. Apparently, here there is also a light-dependent transmembrane
movement of an electron from the excited chlorophyll dimer to QA located on the
opposite side of the hydrophobic barrier, and only one of two ChlII- and pheophytin-containing chains is operative.
64
65
Lys-145 or Lys-222 serve as the ligands for the heme iron. The heme is located in
the large (residues 1250) N-terminal domain exposed to the aqueous phase in the
thylakoid lumen. Residues 251270 are hydrophobic and form an a-helical segment that functions as a membrane-spanning anchor. The remaining 15 C-terminal
amino acids are located on the outer surface of the thylakoid membrane and can be
removed by proteases. Segment 190249 includes many acidic amino acid residues facing the thylakoid interior and is probably involved in interaction with a
positively charged segment of cytochrome b6. Ten conserved basic amino acids in
the region 58154 are assumed to participate in binding the next electron carrier
plastocyanin. The cytochrome f spectrum shows an a-band maximum at 555 nm.
The redox potential of this cytochrome is +365 mV (Cramer et al. 1985).
The average diameter of the b6f complex is about 8.5 nm. In natural membranes
the complexes tend to form dimers (Cramer et al. 1985).
It should be stressed that b6f complex is the slowest and most vulnerable part of
the photosynthetic redox chain. The entrance to the Q-cycle from photosystem 2 is
blocked by the potent herbicide DCMU. Certain steps of the Q-cycle are very
66
2.3.5 Fate of D
lH Generated by the Chloroplast Photosynthetic
Redox Chain
Thylakoids of chloroplasts perform only two functionsreduction of NADP+ and
synthesis of ATP mediated by D
lH generation and consumption. The energy of
D
lH is quantitatively transformed into energy of ATP by the H+-ATP synthase.
The thylakoid does not have to perform osmotic work to transport metabolites,
for the aqueous intrathylakoid space is in fact enzymatically empty. The only
major process that takes place on the inner surface of the thylakoid membrane is
photolysis of water, resulting in formation of molecular oxygen. For both the
substrate (H2O) and product (O2) of this reaction, crossing the membrane poses no
problem. The conversion of ADP and Pi to ATP and of NADP+ to NADPH both
occur on the outer surface of thylakoids.
67
Reverse electron transfer against the redox potential gradient is absent from
thylakoids, which solve the reducing power generation problem by formation of
NADPH in the noncyclic photoredox chain. Such D
lH consumers as H+-motors
rotating bacterial flagella are also absent from thylakoids.
Since there is only one D
lH -linked function in the thylakoid membrane, there
is no need to buffer D
lH as strongly as in bacteria. Nevertheless, thylakoids have
a system that can buffer D
lH to some degree. The thylakoid membrane is rather
permeable to Cl-, K+, and Mg2+. The transport of these ions down the electric
gradient formed by D
lH generators results in the transduction of DW to DpH.
Since the amount of energy equivalent stored in the form of DpH is much larger
than that in DW, the DW ? DpH transition increases the total reserve of membrane-linked energy in the system. This effect should stabilize the rate of ATP
synthesis in spite of light intensity fluctuations occurring in vivo. A general
scheme of energy transduction in the thylakoid membrane is shown in Fig. 2.28.
There is one more essential point that should be mentioned when discussing
chloroplast energetics. It was found that H+-ATP synthase can be absent from
those parts of thylakoids in which the membranes of two adjacent thylakoids
adjoin each other. This occurs when thylakoids are not swollen (i.e. have a discoid
shape) and a very narrow cavity separates one thylakoid from another. In such a
case, all the H+-ATP synthase molecules were shown to be concentrated at the
very periphery of the thylakoid disks exposed to the chloroplast stroma and
(mainly) in membranous lamellas stretched through the stroma (see above,
Fig. 2.20). These lamellas are in fact the continuation of some thylakoids. The
lamellas were shown to contain mainly photosystem 1, whereas thylakoids have
both photosystems. It appears that lamellas specialize in ATP synthesis coupled to
cyclic electron transport around photosystem 1, and they are not capable of
68
photolysis of water and reduction of NADP+ (these two processes occur in thylakoids). Apparently, to form ATP the thylakoid-generated D
lH must be first
transmitted along the membrane to the lamellas, where a major pool of H+-ATP
synthase is located.
When thylakoids are in a swollen state, H+-ATP synthase molecules diffuse
from the lamellas to thylakoids so as to be equally distributed between all the areas
on the intrachloroplast membranes.
All the above-mentioned considerations also apply to the thylakoids of cyanobacteria, which apparently were the evolutionary precursors of chloroplasts. In
this case, however, we must supplement the scheme shown in Fig. 2.27 by a
respiratory chain arranged in the same thylakoid membrane. (In plant cells, the
respiratory chain is located in mitochondria.) The interaction of the photosynthetic
and respiratory chains that share the same components of the Q cycle in their
middle stages will be discussed later (see Sect. 4.3.3).
References
Blankenship RE, Prince RC (1985) Excited state redox potentials and the Z scheme of
photosynthesis. TIBS 10:382383
Clark RD, Hind G (1983) Spectrally distinct cytochrome b-563 components in a chloroplast
cytochrome b-f complex: Interaction with a hydroxyquinoline N-oxide. PNAS 80:62496253
Cramer WA, Widger WR, Herrmann RG, Trebst A (1985) Topography and function of thylakoid
membrane proteins. TIBS 10:125129
Crofts AR, Wraight CA (1983) The electrochemical domain of photosynthesis. Biochim Biophys
Acta 426:149185
Deisenhofer J, Epp O, Miki K, Huber R, Michel H (1984) X-ray structure analysis of a membrane
protein complex. Electron density map at 3 resolution and a model of the chromophores of
the photosynthetic reaction center from Rhodopseudomonas viridis. J Mol Biol 180:385398
Deisenhofer J, Epp O, Miki K, Huber R, Michel H (1985a) Structure of the protein subunits in the
photosynthetic reaction centre of Rhodopseudomonas viridis at 3 resolution. Nature
318:618624
Deisenhofer J, Michel H, Huber R (1985b) The structural basis of photosynthetic light reaction in
bacteria. TIBS 10:243248
Deprez J, Trissl HW, Breton J (1986) Excitation trapping and primary charge stabilization in
Rhodopseudomonas viridis cells, measured electrically with picosecond resolution. PNAS
83:16991703
Drachev LA, Kaulen AD, Ostroumov SA, Skulachev VP (1974) Electrogenesis by bacteriorhodopsin incorporated in a planar phospholipid membrane. FEBS Lett 39:4345
Drachev LA, Kaulen AD, Semenov AY, Severina II, Skulachev VP (1979) Lipid-impregnated
filters as a tool for studying the electric current-generating proteins. Anal Biochem 96:250262
Dracheva SM, Drachev LA, Zaberezhnaya SM, Konstantinov AA, Semenov A, Skulachev VP
(1986) Spectral, redox and kinetic characteristics of high-potential cytochrome c hemes in
Rhodopseudomonas viridis reaction center. FEBS Lett 205:4146
Glaser EG, Crofts AR (1984) A new electrogenic step in the ubiquinol:cytochrome c2
oxidoreductase complex of Rhodopseudomonas sphaeroides. Biochim Biophys Acta 766:
322333
References
69
70
Shuvalov VA, Amesz J, Duysen LNM (1986) Picosecond charge separation upon selective
excitation of the primary electron donor in reaction centers of Phodopseudomonas viridis.
Biochim Biophys Acta 851:327330
Skulachev VP (1972) The driving forces and mechanisms of ion transport through coupling
membranes. FEBS Simposia 28:371385
Skulachev VP (1988) Membrane bioenergetics. Springer, Berlin
Stroebel D, Choquet Y, Popot JL, Picot D (2003) An atypical haem in the cytochrome
b6f complex. Nature 426:413418
Wakao N, Yokoi N, Isoyama N, Hiraishi A, Shimada K, Kobayashi M, Kise H, Iwaki M, Itoh S,
Takaichi S, Sakurai Y (1996) Discovery of natural photosynthesis using Zn-containing
bacteriochlorophyll in an aerobic bacterium Acidiphilium rubrum. Plant Cell Physiol
37:889893
Widger WR, Cramer WA, Herrmann RG, Trebst A (1984) Sequence homology and structural
similarity between cytochrome b of mitochondrial complex III and the chloroplast
b6f complex: position of the cytochrome b hemes in the membrane. PNAS 81:674678
Witt HT (1979) Energy conversion in the functional membrane of photosynthesis. The central
role of the electric field. Biochim Biophys Acta 505:335427
Chapter 3
Organotrophic Energetics
3:1
71
72
3 Organotrophic Energetics
Fig. 3.1 Scheme for unification of fuel (reducing equivalents) used in organotrophic
energetics. Pathways of hydrogen atom transfer are shown in green, reactions of ATP (GTP)
synthesis red, metabolic pathways of fuel unification black, and electron transfer along the
respiratory chain is marked with broad arrows
73
According to Eq. 3.1, full oxidation of one hexose molecule means transfer of
24 electrons from this molecule to six O2 molecules. It is obviously not possible to
perform this within one stage, and that is why this oxidation takes a few consecutive steps. The transfer of an electron to oxygen is coupled to a substantial
release of energy. The conservation of this energy in the form of ATP is a rather
complicated process that requires the functioning of respiratory chain enzyme
complexes. For this to take place, electrons need to enter the respiratory chain. So,
they are first taken from the monomers and transferred to specific cofactor moleculesnicotinamide adenine dinucleotide (NAD+) or (less often) ubiquinone
(CoQ). The NADH and CoQH2 formed during these reactions are oxidized by the
respiratory chain complexes and later by molecular oxygen. So, from the bioenergetic perspective, oxidation of glucose and other compounds and the resulting
unification of reducing equivalents into just two compounds (NADH and CoQH2)
are the main results of the first stages of metabolism of carbohydrates.
Oxidation of glucose usually involves consecutive functioning of two systems:
(i) glycolytic oxidation of glucose to pyruvate and (ii) oxidation of pyruvate to
CO2 in the Krebs cycle (the scheme of these metabolic pathways and photo of
Hans Krebs are presented in Figs. 3.2 and 3.3, respectively). Glucose is first
phosphorylated at the expense of ATP, thus forming glucose-6-phosphate. As
mentioned earlier, cell energy supply is the main function of carbohydrate
metabolism. But already at the first stage the cell needs to spend energy that is
used to form an ester bond between the sugar and a phosphoric acid residue. The
greater part of the energy of ATP cleavage is dissipated as heat, which makes this
process irreversible. This fact is supposed to provide regulation of glycolysis.
Control over any kind of metabolic process is most often realized via regulation of
its first reaction. It seems obvious that such regulation will be simplified if the
regulated process is irreversible. However, there is no ATP consumption when
glycogen, rather than glucose, is used as a substrate. In this case it is inorganic
phosphate that is the phosphoryl source for the reaction of glucose phosphate
formation (the glycogen phosphorolysis reaction is catalyzed by glycogen phosphorylase, where the attachment of phosphate to sugar occurs at the expense of
energy of glucose residue bonds in glycogen).
Glucose-6-phosphate is later isomerized to fructose-6-phosphate. Fructose-6phosphate is again phosphorylated at the expense of ATP, thus forming fructose-1,
6-bisphosphate, i.e., one more glycolytic reaction is accompanied by energy consumption. Again this is most likely needed for regulation of this metabolic pathway,
especially due to the fact that glucose-6-phosphate besides glycolysis can also
participate in the pentose phosphate pathway or be used for synthesis of glycogen
and other carbohydrates. So, it is no wonder that the main control over glycolysis
takes place at the stages that are catalyzed by hexokinase and phosphofructokinase.
In the next stage, fructose-1,6-bisphosphate is cleaved to 3-phosphoglyceric
aldehyde and dihydroxyacetone phosphate. The latter is then converted to phosphoglyceric aldehyde (PGA). The aldehyde is in turn oxidized to 3-phosphoglyceric
acid (PG). The first oxidative stage of the glucose metabolism pathway occurs just
here. As a result, two electrons are transferred from each of the two parts of the
74
3 Organotrophic Energetics
Fig. 3.2 Main pathway of aerobic glucose catabolism: a oxidation of glucose to pyruvate;
b pyruvate oxidation in the Krebs cycle. Under anaerobic conditions, pyruvate is reduced by
NADH to lactate (glycolysis; see Fig. 3.6)
Fig. 3.3 Hans Krebs
75
acid to form citric acid, which is then isomerized to isocitric acid. The latter
undergoes oxidative decarboxylation (which is accompanied by electron transfer
to NAD+), thus forming a-ketoglutaric acid. The a-ketoglutarate undergoes oxidative decarboxylation once again, this process being accompanied by electron
transfer to NAD+, and it turns into succinic acid (this process takes two consecutive steps). Succinic acid is later oxidized to fumaric acid. But the redox potential
of the succinate/fumarate couple (+30 mV) is much more positive than the
potential of the NADH/NAD+ couple (-320 mV), and in this case electrons from
the organic substrate are transferred not to NAD+, but to an electron carrier with a
more positive redox potentialubiquinone (CoQ, +60 mV).
Fumaric acid is hydrated to form malic acid, which is in turn oxidized (with
electrons being transferred to NAD+) to oxaloacetic acid, which is ready for the
condensation reaction with the next acetyl-CoA molecule.
So, we can see that during glycolysis and the Krebs cycle full oxidation of a
glucose molecule to carbon dioxide occurs, and electrons taken from the substrate
are used for formation of NADH and ubiquinol (CoQH2):
C6 H12 O6 10 NAD 2 CoQ 6 H2 O
! 6 CO2 2 CoQH2 10 NADH 10 H
3:2
The NADH and CoQH2 can be later oxidized by molecular oxygen via the
respiratory chain:
10NADH 10 H 2 CoQH2 6 O2
! 10 NAD 2 CoQ 12 H2 O
3:3
76
3 Organotrophic Energetics
77
78
3 Organotrophic Energetics
Fig. 3.5 Oxidative decarboxylation of pyruvate and a-ketoglutarate. These ketoacids are shown
in green; high-energy bond is in red
79
80
3 Organotrophic Energetics
short but intensive exercise. In glycolysis, glucose is converted into two molecules
of pyruvic acid. Two ATP molecules (they are the only source of energy for
lactate-producing bacteria) and two NADH molecules are also formed. The latter
must be oxidized for further glycolytic reactions to proceed. In the case of lactic
acid fermentation, oxidation of NADH is achieved by reduction of pyruvate to
lactic acid. The general scheme of lactic acid fermentation is presented in Fig. 3.6.
This scheme also shows the importance of maintaining the complete redox balance
of fermentation. In the case of a compound more reduced compared to glucose
(e.g. sorbitol) or more oxidized (e.g. gluconic acid), it is no longer possible to keep
that balance. That is why these compounds that seem so similar to glucose are not
fermentable substrates for lactic acid-producing bacteria.
Let us try to answer the question of where the energy necessary for ATP
synthesis in the course of lactic acid fermentation comes from. When considering
glycolysis, it is necessary to note that all the carbon atoms in a glucose molecule
have an intermediate degree of reduction (all of them are connected to a hydroxyl
or belong to a carbonyl group). Reduction of certain carbon atoms of glucose due
to oxidation of other atoms of the same sugar molecule is energetically profitable.
This happens when glucose is converted into lactate, where the first carbon atom is
oxidized due to the reduction of the third carbon atom.
So, two ATP molecules are formed per consumed glucose molecule during
glycolysis. Similar stoichiometry is characteristic also for the well-known fermentation yielding ethanol. Is there a way to increase ATP/glucose stoichiometry
81
of fermentation? In fact, it is possible. One only needs to increase electron disproportionation between different carbon atoms of the fermentation products. Let
us consider this phenomenon using mixed fermentation of enterobacteria as an
example. It has already been described that there are two pyruvate molecules, two
NADH molecules, and two ATP molecules formed per metabolized glucose
molecule decomposed using the canonical glycolytic pathway. It would seem very
tempting not to reduce pyruvate to lactate but to obtain acetyl*CoA and later
acetate and ATP instead (the way it has already been described for the mechanism
of oxidative decarboxylation of pyruvate). Then we would obtain four ATP
molecules per glucose consumed. But in this case it would not be possible to
oxidize two NADH molecules that have been formed in the course of glycolysis.
Even more so, two additional NADH molecules are formed during the functioning
of the pyruvate dehydrogenase complex, thus making it impossible to maintain the
redox balance of fermentation. That is why enterobacteria chose a different path.
They use pyruvate-formate lyase instead of pyruvate dehydrogenase (Fig. 3.7)
(Knappe and Sawers 1990). This enzyme forms acetyl*CoA and formate from
pyruvate (and not acetyl*CoA, CO2, and NADH as in the case of the pyruvate
dehydrogenase reaction). The further fates of the two acetyl*CoA molecules
formed from one glucose are different. One undergoes phosphorolysis, thus
forming ATP and acetate. The second acetyl*CoA molecule is reduced twice by
NADH (complete oxidation of NADH obtained during glycolysis occurs), thus
forming ethanol and free CoA. The described process leads to the synthesis of
three, rather than two, ATP molecules.
And what would the ideal (from the perspective of energetic output) fermentation look like? It would most likely mean the maximum possible electron disproportionation between the different carbon atoms in the fermentation products,
i.e., the following process:
C6 H12 O6 ! 3 CO2 3 CH4
3:4
82
3 Organotrophic Energetics
3.5 Carnosine
The very low energetic outcome of fermentation seems to be the main drawback of
this type of metabolism. It means that a large amount of fermentable substrate
needs to be consumed by the cell to be supplied with a sufficient amount of energy,
and this will lead to a substantial accumulation of fermentation products, some of
which can be toxic. In many cases of anaerobic fermentation, such products are
lactic, acetic, succinic, propionic, or butyric acids which completely dissociate to
corresponding anions and H+ ions at neutral pH.
This fact is of particular importance in the case of intense work of skeletal
muscles. The muscles exhaust oxygen under these conditions, and a substantial
amount of lactic acid accumulates. This acidifies the cytoplasm, and muscle
enzymes can be inactivated by excess H+ ions. To consider how an organism
solves this problem we will discuss muscle dipeptides. Peptide history is an
interesting chapter in biochemistry. Peptides regulate different processes in our
body, and they are very important components of our body. Now, very few people
remember that the first peptide was discovered in Russia at the turn of the nineteenth and twentieth centuriesin 1900by Vladimir Gulevitch (Fig. 3.8). This
compound, b-alanyl-histidine (Fig. 3.9), was given the name carnosine (Gulevitch
and Amiradzhibi 1900). Later it was found that carnosine has an analog where one
of the hydrogen atoms in the histidine heterocycle is substituted by a methyl group
(b-alanyl-methylhistidine, or anserine). These two compounds are present in many
muscles in very large amounts. Their total concentration in cytoplasm sometimes
reaches 50 mM.
At the very beginning of the twentieth century, 30 years before the discovery of
ATP, many metabolites were unknown, and new compounds of biological origin
were discovered every year. With the passing of time, the role of the majority of
new compounds became clear, but the functions of carnosine and anserine
remained unknown. At the end of his life path, Gulevitch bequeathed the study of
these compounds to his beloved student, Sergey Severin (Fig. 3.10). Severin
remained true to his professors wishhe dedicated his whole life to the discovery
of the physiological role of carnosine and anserine. Many years after Gulevitchs
death, in 1953, the function of muscle dipeptides was clarified by his former
student. But this function proved to be so simple and apparently so primitive that
Severin in fact could not believe that he solved the problem formulated by his
mentor. Later, up to the end of his long life (Severin lived for more than 90 years),
he continued to look for an answer to the question that had been already
answeredwhat is the physiological role of carnosine and anserine. Let us look at
the experiment that became known in physiology as the Severin phenomenon.
An isolated frog muscle was stimulated by an electrode and began to contract.
After some time, the muscle became tired and contraction ceased. However, when
carnosine was added to the solution, the muscle was able to work for hours without
any traces of fatigue (Severin et al. 1953). This was a truly spectacular effect.
Severins coworkers invested much effort in attempts to understand the mechanism
3.5 Carnosine
83
of this phenomenon; they measured many different parameters, and it was shown
that nothing was changed by carnosine besides one thingthe muscle was able to
accumulate very high amounts of lactate if carnosine was added. A simple
84
3 Organotrophic Energetics
3.5 Carnosine
85
their biological function (recall the fact that the great majority of enzymes have a
pH optimum for activity).
Ultimately, the pH-buffering function of carnosine turned out to be not its only
function (Boldyrev 2000). Carnosine is also a good chelator of copper and iron
ions, this fact being of substantial importance for cells, as these cations catalyze
the transformation of hydrogen peroxide to OH radical, one of the strongest
metabolic poisons (see Chap. 15). Carnosine also protects proteins and DNA from
the destructive effects of aldehydes. High concentrations of carnosine probably
protect us from intoxication caused by formaldehyde and other aldehydes. Aldehydes are sure to damage carnosine, but it is rather cheapit is just a dipeptide.
Carnosine also protects cells from accumulation of amyloid fragments, from
undesirable proteinprotein interactions, etc. But all these functions are secondary,
as they do not explain why it was dipeptides with their ability to buffer the H+ level
near pH 7 that were chosen, and why it is only in muscles that they are present in
very high concentrations.
References
Bate Smith EC (1938) The buffering of muscle in rigor; protein, phosphate and carnosine.
J Physiol 92:336343
Boldyrev AA (2000) Problems and perspectives in studying the biological role of carnosine.
Biochemistry (Mosc) 65:751756
Deutsch A, Eggleton P (1938) The titration constants of anserine, carnosine and some related
compounds. Biochem J 32:209211
Effron MB, Guarnieri T, Frederiksen JW, Greene HL, Weisfeldt ML (1978) Effect of
tris(hydroxymethyl)aminomethane on ischemic myocardium. Am J Physiol 235:H167H174
Gulevitch VS, Amiradzhibi S (1900) Ueber das Carnosin, Eine Neue Organische Base des
Fleischextractes. Ber Deutsch Chem Ges 33:19021903
Junge W, McLaughlin S (1987) The role of fixed and mobile buffers in the kinetics of proton
movement. Biochim Biophys Acta 890:15
Knappe J, Sawers G (1990) A radical-chemical route to acetyl-CoA: the anaerobically induced
pyruvate formate-lyase system of Escherichia coli. FEMS Microbiol Rev 6:383398
Severin SE, Kirzon MV, Kaftanova TM (1953) Effect of carnosine and anserine on action of
isolated frog muscles. Dokl Akad Nauk SSSR 91:691701 (In Russian)
Chapter 4
The term respiratory chain applies to a reaction sequence responsible for the
transfer of hydrogen atoms or electrons from respiratory substrates to molecular
oxygen.
There are two types of respiratory chainscoupled to energy transduction and
noncoupled. The biological significance of the first type was recognized in the
early 1930s when Vladimir Engelhardt (Fig. 4.1) discovered respiratory phosphorylation (Engelhardt 1930). The role of the second type became clear almost
30 years later. In 1960, one of this books authors (V.P.S.) suggested that
uncoupled respiration can be biologically useful, e.g., under conditions when heat,
rather than ATP, becomes the most important for organisms (Skulachev and
Maslov 1960).
This chapter describes the energy transduction-coupled respiratory chain in
mitochondria and in certain bacteria. In the case of eukaryotic cells, respiration
coupled to energy transformation is localized in the inner mitochondrial membrane. In the case of respiring bacteria, the process can be found in the cytoplasmic
membrane, mesosomes, or thylakoids.
87
88
Heterotrophic organisms are usually not capable of direct use of light energy. In
fact, they utilize the energy that has been accumulated by photosynthesizing
plants. Roughly speaking, heterotrophic organisms reverse the reactions of photosynthesis. They first oxidize carbon atoms of carbohydrates (as well as of fatty
acids and amino acids) to CO2 (as described in Chap. 3) and use the electrons
obtained for reduction of NAD+. The NADH formed is then oxidized by molecular
oxygen via the respiratory chain so that water is formed. It has already been
mentioned that a very large amount of free energy (about 1.1 electron volt per
electron transferred from NADH to oxygen) is released when electrons are
transferred from NADH to O2. This energy can be stored by the respiratory chain
in the form of D
lH . The proton-motive force on biological membranes is usually
about 200 mV (if the electric potential reaches higher values, it can lead to disruption of the lipid bilayer and the loss of the stored energy). Calculation shows
that transfer of four electrons from two NADH molecules to O2 needs to be
coupled to translocation of at least 20 protons across the membrane for the energy
to be stored in the form of D
lH . It is the necessity of such a high H+/O2 stoichiometry that was most likely the reason for Nature to have chosen a complicated
mechanism of energy conservation when NADH oxidation was split into three
discrete energy-releasing steps. Electrons are first transferred from NADH to
ubiquinone by NADH: CoQ-oxidoreductase (complex I), the ubiquinol obtained is
then oxidized by cytochrome c via CoQH2:cyrochrome c-oxidoreductase (also
called bc1 complex or complex III), and finally the reduced cytochrome c is
oxidized by molecular oxygen via cytochrome c oxidase (complex IV). The
activity of all of these enzyme complexes is coupled to generation of proton
potential that can be utilized to form ATP. In this context, it should be indicated
89
that Albert L. Lehninger and David E. Green (Fig. 4.2) made a crucial contribution
to discovery of energy coupling sites in the respiratory chain and purification of the
respiratory chain complexes responsible for energy coupling.
The redox potential difference between the substrates and products of the
reaction is *380 mV for complex I, *190 mV for complex III, and *570 mV
for complex IV (see Fig. 4.3).1 If we assume the value of D
lH equal to
*200 mV, we can calculate that complexes I, III, and IV can transfer 2, 1, and
23 protons, respectively, per electron transported. Experiments have shown the
H+/e ratios equal to 2, 1, and 2 for complexes I, III, and IV, respectively
(Wikstrm 1977, 1984; Galkin et al. 1999).
Comparison of potentials and H+/e stoichiometry makes it obvious that complexes I and III store almost all the energy in the form of D
lH released during
electron transfer. The energy released at the last (cytochrome oxidase) step of the
respiratory chain is substantially higher than at the previous spans of the chain.
Nevertheless, H+/e stoichiometry for cytochrome oxidase is the same as for
complex I (two H+ per one e). So, a substantial part of the energy is dissipated in
the form of heat at the last stage of respiration, and in contrast to the reactions of
the previous stages, this stage is irreversible. In fact, huge energy drop
It is important to note that it is the standard redox potentials (E00 ) that are given both in the text
and in Fig. 4.3. The real values of redox potentials (E) in a cell can differ substantially from those
E00 values because of the difference in concentrations of corresponding oxidized and reduced
forms of the redox compounds involved.
90
91
the terminal segment of the respiratory chain. This is the case for a Krebs cycle
substrate, succinate (redox potential, +30 mV) as well as for fatty acyl-CoA (redox
potential, about -10 mV), the substrate of the first oxidoreduction in the fatty acid
b-oxidation system (Sato et al. 1999). Succinate dehydrogenase and acyl-CoA
92
Fig. 4.5 Redox potential scale of different compounds related to respiratory chain
93
2
In the case of enterobacteria, NDH-1 consists of 13 subunitsnot because of the lack of one of
the main polypeptides, but rather due to fusion of the genes of two subunits (nuoC and nuoD) into
one elongated gene (nuoCD).
94
Fig. 4.6 Spatial arrangement of complex I from the bacterium Escherichia coli (peripheral
domain is shown yellow, and membrane domain blue) and complex I from the mitochondria of
the fungus Neurospora crassa (light-blue shading) (Guenebaut et al. 1998)
in the cytoplasm, and enter mitochondria after being transported across the two
mitochondrial membranes.3
Complex I was shown to be L-shaped being composed of two domains with a right
angle between them (Fig. 4.6). One of the domains is located inside the membrane,
and the other peripheral domain protrudes by about 150 from the membrane into
the mitochondrial matrix (or cytoplasm in the case of bacteria). The majority of the
redox centers of the enzyme are located in this peripheral domain.
We note that this principle (i.e. the fact that the mitochondrial genome possesses only the
genes of the most hydrophobic subunits) is characteristic also for other respiratory chain
components. Transport of such hydrophobic polypeptides through the cytoplasm and across the
outer mitochondrial membrane would be very difficult. That is probably why the corresponding
genes were left in the mitochondrial genome instead of being transferred together with the vast
majority of other mitochondrial protein genes into the nucleus. This seems to be a likely
explanation of why this unique organelle has kept its own genome.
95
HP
IP
IP
IP
FP
FP
HP
19
28
25
65
18
45
90
NuoH
NuoI
NuoJ
NuoK
NuoL
NuoM
NuoN
HP
IP
IP
HP
HP
HP
HP
32
23
23
10
45
40
38
[4Fe4S] (N2)
[2Fe2S] (N1a)
NADH, FMN, [4Fe4S] (N3)
[4Fe4S] (N4), [4Fe4S] (N5),
[2Fe2S] (N1b)
[4Fe4S] (N6a), [4Fe4S] (N6b)
96
proteins has shown that the main part of the IP subfragment originates from watersoluble [NiFe] hydrogenase which catalyzes electron transfer to H+ ions, thus
forming molecular hydrogen. The membrane part of complex I (HP) has certain
similarities to the multisubunit complex Mrp of alkalophilic bacilli, which is
supposed to function as a Na+(K+)/H+-antiporter (Efremov and Sazanov 2011).
Unification of these two enzymatic modules probably made it possible to create a
protein capable of coupling electron transfer from FeS clusters to quinone and
transmembrane proton transport. One more part of complex I, subcomplex FP,
functions as an adaptor and provides substrate specificity of this enzyme to its
electron donor. It is interesting to note that a number of prokaryotes have enzymes
homologous to complex I that contain some other subunits nonhomologous to
NuoE and NuoF instead of subcomplex FP. This replacement led to a change in
substrate specificity. For instance, in some archaea a similar enzymatic complex
oxidizes cofactor F420H2 instead of NADH, in cyanobacteria and chloroplasts it
oxidizes NADPH, and in the bacterium Helicobacter pylori (which causes gastric
ulcer in humans) it uses ferredoxin as the reductant.
97
must be mentioned that rotenone is as much a poison for other animals as it is for
fish. Capsaicin is another interesting example of complex I inhibitors. This compound is responsible for the burning taste of red pepper. Capsaicin inhibits complex I only at relatively high concentrations, but all the known representatives of
this group of enzymes are sensitive to this compound (Yagi 1990). That is why
capsaicin is often used for identification of complex I in respiratory chains of
different organisms. It is important to note that many detergents (including those
often used in everyday life) are strong complex I inhibitors, and thus they are quite
dangerous. For instance, dodecyl sulfate and Triton X-100 act in this manner
(Grivennikova et al. 2003).
98
99
4
Complex I from Thermus thermophilus, when compared to other homologous proteins,
contains an extra [4Fe4S] cluster (N7). But this cofactor is not conservative. It seems not to
participate in electron transfer from NADH to quinone.
100
101
102
(Fig. 4.9c, d) (Efremov et al. 2010; Efremov and Sazanov 2011). A similar but
somewhat shorter horizontal a-helix was observed in the three-dimensional
structure of mitochondrial complex I from Y. lipolytica (Hunte et al. 2010).
A piston model was proposed to explain how the energy might be transferred
over a long distance in the membrane domain (Efremov et al. 2010). Delivery of
electrons from NADH to quinone was postulated to cause conformational changes
in the proximal membrane subunits leading to movements of the amphipathic
a-helix, which is in contact with transmembrane helices. Such movements
occurring along the membrane might result in formation of proton-conducting
channels in the NuoL, M, and N subunits and, as a consequence, in transmembrane
proton translocation. In this hypothesis, the amphipathic a-helix plays the role of a
mechanical piston (analogs to the piston of a steam engine) that propagates longdistance conformational changes and results in proton pumping (Efremov and
Sazanov 2011). Further studies are needed to test this hypothesis.
103
Function
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
Unknown
Unknown
Binds hemes bL and bH
Binds heme c1
Binds [2Fe2S] cluster
Binds CoQ
Unknown
Unknown
Unknown
Binds cytochrome c
Binds Rieske protein
49.5
47.0
44.0
28.0
21.5
13.5
9.5
9.0
8.0
7.0
6.5
Core protein I
Core protein II
cytochrome b
cytochrome c1
Rieske protein
QIII protein
104
complex seemed to be supported by their size (they are larger than other complex
subunits) and by their lack of redox groups. Later these proteins were found to be
peripheral. The smallest polypeptide of complex III (subunit XI) participates in the
binding of Rieske protein. Functions of the other polypeptides with no redox
centers remain unclear.
Only one of 11 polypeptides that constitute the bc1 complex of eukaryotes,
namely cytochrome b, is coded in the mitochondrial genome. The genes of the
other proteins are in the cell nucleus.
It is noteworthy that the respiratory chain of many bacteria also contains an
enzyme homologous to the bc1 complex of eukaryotes but like the bacterial H+translocating NADH:quinone-oxidoreductases, prokaryotic bc1 complexes have
far simpler structure than their mitochondrial analogs. Bacterial enzymes consist
of just three polypeptides homologous to three catalytic subunits of mitochondrial
complex III (cytochrome b, cytochrome c1, and Rieske protein).
It has been already discussed (see Sect. 2.3.3) that a very close analog of bc1
complex, namely b6f complex, functions in plant chloroplasts. It participates in
operation of the photosynthetic redox chain, where it transfers electrons from
photosystem II to photosystem I. Considering its mechanism of functioning,
b6f complex is practically a full analog of mitochondrial complex III. It is interesting to note though that b6f complex contains a number of additional prosthetic
groups. The fact that it has three instead of two hemes is the most interesting
difference. This additional heme, which was coined ci, is located next to the bH
heme (Zhang et al. 2004). Its function is still unclear.
105
Fig. 4.11 Three-dimensional structure of yeast complex III. a Homodimer complex that is
composed of catalytic subunits of cytochrome b (blue), Rieske protein (green), and cytochrome
c1 (yellow) with the corresponding prosthetic groups (black) and also of eight additional subunits,
six of which are indicated by separate colors: core protein I (purple), core protein II (blue-green),
subunit VI (blue), VII (red), VIII (pink), and IX (gray). b Catalytic subunits of one monomer. QP
and QNquinone binding sites in complex III (Hunte et al. 2003)
The X-ray analysis also helped to reveal the two CoQ-binding sites in the bc1
complex. They were given the names P and N centers (P) and (N) are for positively
and negatively charged compartments of the mitochondrion, respectively. Other
names are o (out) and i (in) centers.
Figure 4.12 presents the distances between the electron transport centers of
complex III. One can see that the arrangement makes it possible to transfer
electrons not just within a certain monomer, but also between monomers of the
homodimer of this complex (Hunte et al. 2000).
106
107
108
109
4.4.1 Cytochrome c
Cytochrome c localized in the mitochondrial intermembrane space functions as a
link in the process of reducing equivalent transfer from complex III to complex IV.
This water-soluble protein with molecular mass of 12 kDa is composed of 104
amino acid residues. The spatial structure of cytochrome c was established on the
basis of X-ray analysis with the resolution of 1.5 (Fig. 4.13). This electron
carrier seemed to have been studied quite thoroughly. The more amazing then was
the discovery made in the last decade of the twentieth century. Cytochrome c was
shown to be one of the death proteins that participate in a cell suicide (apoptosis). We will discuss this phenomenon in one of the last chapters of this book
(Chap. 15), and here we will concentrate mainly on the vital function of cytochrome c as the respiratory chain electron carrier.
The His-18 and Met-80 residues play an important role in the functioning of
cytochrome c because they are the axial ligands of the iron atom in the heme.
Similar to all the analogous cytochromes of C type, mesoheme C is covalently
bound to sulfhydryl groups of cysteine residues of the protein (Cys-14 and Cys17). This is the difference between the c-type cytochromes and those of other types
(a, b, d, o, and other), where hemes are connected in a noncovalent manner. The
covalent binding of heme C to apocytochromes was suggested to be the result of
the fact that majority of the c-type cytochromes are located on the outer side of the
mitochondrial or bacterial membrane (Wood 1983). A noncovalently bound heme
might be dissociated and diluted in the cytosol (in case of mitochondria) or periplasm and then the outer medium (in the case of bacteria).
The three-dimensional structure of cytochrome c is reminiscent of a mitten that
keeps the heme in such a position that the heme edge reach to the surface of the
protein (Takano and Dickerson 1980). A corona of positively charged amino acid
residues (Lys-13, Lys-72, Lys-79, and Lys-86) is located close to the heme edge.
Lysine residues are needed to tack cytochrome c to its partnerscytochrome c1
and cytochrome oxidase. For instance, cytochrome oxidase has three negatively
charged amino acids, which (similar to a magnetic lock) provide the electrostatic
attraction of cytochrome c and its fixation in the right position. Oxidation and
reduction of cytochrome c are not accompanied by substantial changes in its
conformation.
The amino acid sequence of cytochrome c was established long before the first
attempts for its X-ray analysis were made. Extraordinary stability, small size, and
relative simplicity of cytochrome c purification were reasons for the rapid accumulation of information on the structure of this protein in different organisms.
When comparing amino acid sequences of cytochromes c extracted from different
sources, genosystematics began to develop their first evolutionary trees. It was
found that humans and chimpanzees have identical primary structure of cytochrome c. When compared to human cytochrome c, rhesus macaques have one
amino acid change, horses 12, chickens 13, tuna 21, wheat 35, and the fungus
Neurospora 44 changes.
110
111
iron are called low-spin (for they have an equilibrium electron configuration that
corresponds to the lowest energy level with the minimal spin s = 1/2). Such hemes
usually function as electron carriers; all the cytochromes described in the previous
chapters as well as heme a of cytochrome oxidase belong to this group. Nevertheless, certain hemes (e.g. heme a3) lack one of the axial ligands. The spin of such
hemes is 5/2, so they are called high-spin. When small nucleophilic compounds are
present in the medium (e.g. O2, NO, CO, H2S, HCN, azide, etc.) they can bind to a
high-spin heme, thus operating as its second axial ligand. In the case of oxygen,
such bonding can be accompanied by electron transfer from the heme to O2, and
hence oxygen-reductase activity will occur. So, high-spin hemes can function not
only as electron carriers, but also as the binding sites for small ligands, (like
oxygen in myoglobin and hemoglobin, or NO in guanylate cyclase), or as sites of
their reduction (e.g. oxygen reduction in cytochrome oxidase or NO reduction in
NO-reductase).
112
Fig. 4.15 Structure of K- and D-channels in bovine cytochrome oxidase. Small light-blue
spheres represent water molecules
113
Fig. 4.16 A six step scheme of the process of reduction of molecular oxygen in the binuclear
center of cytochrome c oxidase. Ypresumably tyrosine-244 of subunit I
Figures 4.14 and 4.15 show these centers to be located approximately along one
line parallel to the membrane plane (the hemes themselves are located perpendicular to the membrane); they are closer to its external surface (about 15 away
from this surface). The X-ray analysis studies showed an interesting feature of the
arrangement of the binuclear cytochrome oxidase center. One of the histidine
residues (which serves as copper CuB ligands), His-240, forms a covalent bond
with the phenol ring of a nearby tyrosine residue, Tyr-244. This covalent bond was
later found to be formed autocatalytically during the turnover of the enzyme. Such
type of posttranslational modification is extremely rare and has been found only in
oxidases so far.
Analysis of the three-dimensional structure of complex IV also suggests the
existence of two channels for proton transport from the mitochondrial matrix to the
binuclear center (see Fig. 4.15). These pathways consist of conservative residues
of polar amino acids and firmly bound water molecules. They were coined as
K- and D-channels according to the abbreviations of amino acid residues located at
the entrance to these pathways, Lys-354 and Asp-124, respectively.
Analysis of the three-dimensional structure of cytochrome oxidase fails to
suggest a pathway(s) of proton transfer from the binuclear center to the intermembrane space. However, this part of the protein is not so hydrophobic; the
distance between the binuclear center and the outer surface of the membrane is
only 15 , so it seems possible that proton transfer does not require any special
channel in this case.
114
is transferred from CuA to the a heme and further to the binuclear hemecopper
[heme a3CuB] center, the terminal respiratory chain component that reduces O2:
4e
4e
4e
4eO2 4H
!
2 H2 O
4:1
However, this equation does not seem to be sufficient for the description of
electron transfer in cytochrome oxidase. Let us remember that four electrons are
needed to reduce an O2 molecule to water, while cytochrome c functions as a
carrier for just one electron. This means that a special mechanism is needed to
accumulate electrons on the oxidase and to reduce O2. Let us look at this mechanism in more detail (Fig. 4.16):
I The oxidation of the first cytochrome c molecule leads to an electron transfer
from cytochrome c to CuA and further to a, a3, and CuB. The enzyme changes
from an oxidized form (O) to a one-electron reduced form (E). This form (E) is
not yet capable of binding oxygen. The exact localization of the first electron on
the enzyme in state E has not been established; we will assume the binuclear
enzyme center to be in the state (Fe3+Cu1+) so as to simplify the discussion.
II Oxidation of the second cytochrome c molecule actuates the following chain of
events:
(a) both electrons (the first and the second) can move to the binuclear center and
reduce it, thus generating the Fe2+Cu1+ form of the enzyme, which was given
the name R;
(b) due to the transfer of the two electrons to the binuclear center, it becomes able
to bind oxygen; so, the R form changes into the A form (the state of the
binuclear center is Fe2+-O2 Cu1+);
(c) oxygen binding is followed by electron transfer from cytochrome oxidase to
this oxygen.
It was first suggested that this stage involved two-electron reduction of O2
leading to peroxide bridge formation between the atoms of iron and copper of
the binuclear center (Fe3+-OO-Cu2+). This form was given the name P, or
peroxide. However, it was later found that it was already at this stage that fourelectron reduction of oxygen was taking place (but this form was still left with
the name P for a historical reason). There surely appears the question how can
this four-electron reduction of oxygen be possible by the 2e-reduced enzyme?
One additional electron for reduction of oxygen was found to be taken from
the heme a3 iron, thus transforming it into the very unusual ferryl state Fe4+.
The other electron (and most likely a proton as well) was found to be transferred from some aromatic amino acid residue located nearby, this process
leading to formation of the radical form of the residue (Y).5 Also, one H+ ion
The radical form Y is considered to be formed from the Tyr-244 residue. Generation of such
an unusual and extremely reactive radical is probably the reason for formation of the covalent
bond between Tyr-244 and a nearby His-240. This covalent bond probably stabilizes the radical
form, which helps to avoid destruction of the protein.
5
115
is taken up, and copper is bound to a hydroxyl anion. So, it now seems that the
P form of cytochrome oxidase looks as follows: Y Fe4+ = O2 -HO-Cu2+. It
should be noted that the redox potentials of the Y/Y and Fe4+/Fe3+ couples are
extremely high (C ? 0.7 V).
So, it is already at this stage that the reduction of oxygen to water occurs. Later
the enzyme only needs to be returned to its initial state. This happens during
the oxidation of the next two cytochrome c molecules.
III During the oxidation of the third cytochrome c molecule, an electron is
transferred to the Y and another proton is taken from the water phase. This
leads to the formation of the ferryl (F) form of the enzyme (Fe4+ = O2 Cu2+)
and the first H2O molecule.
IV The oxidation of the fourth cytochrome c molecule leads to the formation of
the second H2O molecule and regeneration of the original oxidized (O) form of
the enzyme (Fe3+ Cu2+), hence the catalytic cycle of cytochrome oxidase is
completed.
4.4.5 Mechanism of D
lH Generation by Cytochrome c Oxidase
It has been already mentioned that cytochrome oxidase generates D
lH with
stoichiometry of 2 H+/e. When describing the previous components of the respiratory chain, we postulated the existence of two fundamentally different
116
References
117
References
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mitochondrial myopathy due to complex III deficiency with vitamins K3 and C: A 31P-NMR
follow-up study. Annal Neurology 19:598602
Belevich I, Verkhovsky MI (2008) Molecular mechanism of proton translocation by cytochrome
c oxidase. Antioxid Redox Signal 10:129
Bloch D, Belevich I, Jasaitis A, Ribacka C, Puustinen A, Verkhovsky MI, Wikstrm M (2004)
The catalytic cycle of cytochrome c oxidase is not the sum of its two halves. Proc Natl Acad
Sci USA 101:529533
Efremov RG, Sazanov LA (2011) Structure of the membrane domain of respiratory complex I.
Nature 476:414420
Efremov RG, Baradaran R, Sazanov LA (2010) The architecture of respiratory complex I. Nature
465:441445
Eleff S, Kennaway NG, Buist NR, Darley-Usmar VM, Capaldi RA, Bank WJ, Chance B (1984)
31
P NMR study of improvement in oxidative phosphorylation by vitamins K3 and C in a
patient with a defect in electron transport at complex III in skeletal muscle. Proc Natl Acad
Sci USA 81:35293533
Engelhardt WA (1930) Ortho- und pyrophosphate im Aeroben und Anaeroben Stoffwechsel der
Blutzellen. Biochem Z 251:1621
Friedrich T, Scheide D (2000) The respiratory complex I of bacteria, archaea and eukarya and its
module common with membrane-bound multisubunit hydrogenases. FEBS Lett 479:15
Galante YM, Hatefi Y (1978) Resolution of complex I and isolation of NADH dehydrogenase and
an iron-sulfur protein. Methods Enzymol 53:1521
Galkin AS, Grivennikova VG, Vinogradov AD (1999) H+/2e- stoichiometry in NADH-quinone
reductase reactions catalyzed by bovine heart submitochondrial particles. FEBS Lett
451:157161
Grivennikova VG, Ushakova AV, Cecchini G, Vinogradov AD (2003) Unidirectional effect of
lauryl sulfate on the reversible NADH:ubiquinone oxidoreductase (Complex I). FEBS Lett
549:3942
Gunebaut V, Schlitt A, Weiss H, Leonard K, Friedrich T (1998) Consistent structure between
bacterial and mitochondrial NADH:ubiquinone oxidoreductase (complex I). J Mol Biol
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Hirst J, Carroll J, Fearnley IM, Shannon RJ, Walker JE (2003) The nuclear encoded subunits of
complex I from bovine heart mitochondria. Biochim Biophys Acta 1604:135150
Hunte C, Koepke J, Lange C, Rossmanith T, Michel H (2000) Structure at 2.3 resolution of the
cytochrome bc1 complex from the yeast Saccharomyces cerevisiae co-crystallized with an
antibody Fv fragment. Structure 8:669684
Hunte C, Palsdottir H, Trumpower BL (2003) Protonmotive pathways and mechanisms in the
cytochrome bc1 complex. FEBS Lett 545:3946
Hunte C, Zickermann V, Brandt U (2010) Functional modules and structural basis of
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Iwata S, Ostermeier C, Ludwig B, Michel H (1995) Structure at 2.8 resolution of cytochrome
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Leif H, Weidner U, Berger A, Spehr V, Braun M, van Heek P, Friedrich T, Ohnishi T, Weiss H
(1993) Escherichia coli NADH dehydrogenase I, a minimal form of the mitochondrial
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118
Chapter 5
In the previous chapter, we discussed the structure of the respiratory chain from
the mitochondria of higher animals. Relatively high efficiency of energy conservation (high value of H+/O2 coefficient) is characteristic for this type of respiratory
chain, and the electron transfer pathway from NADH to oxygen is linear in this
case. The structure of bacterial and archaeal respiratory chains and also that of
protozoan, plant, and fungal mitochondria have a number of crucial differences
from the electron transport chain of animal mitochondria.
(1) Each of three canonical respiratory chain energy coupling sites (complexes
I, III, and IV) can be bypassed via alternative noncoupled or partially coupled
electron transfer pathways.
(2) Certain enzymes of the bacterial respiratory chains use sodium ions instead of
protons as the primary coupling ions (we will discuss this phenomenon in
more detail in Chap. 12).
(3) Bacteria are capable of utilizing a far broader list of compounds both as
respiratory chain electron donors and acceptors. For instance, many microorganisms, besides oxygen, can reduce such compounds as NO3, NO2, NO,
N2O, different organic S-oxides and N-oxides, fumarate, etc. Along with
NADH, succinate, fatty acids, and glycerophosphate, bacterial respiratory
chains can directly oxidize molecular hydrogen, formate, malate, lactate,
glucose, certain amino acids, Fe2+, NO2, S2O32, etc.
(4) Respiratory chain composition can vary depending on the growth conditions,
and in the case of multicellular organisms it can also be tissue-specific.
These differences are probably connected to different conditions of functioning
of respiratory chains of animal mitochondria as compared to bacterial respiratory
chains and mitochondria of protozoa, fungi, and plants. Animal mitochondria
function under constant conditions of their external medium, i.e., cytosol of these
cells. In contrast, conditions of the functioning of mitochondria of protozoa, plants,
and unicellular fungi, and especially conditions of bacterial growth can undergo
considerable changes within relatively short periods of time. That is why respiratory chain structure of these organisms is far more complex and can vary greatly
V. P. Skulachev et al., Principles of Bioenergetics,
DOI: 10.1007/978-3-642-33430-6_5, Springer-Verlag Berlin Heidelberg 2013
119
120
121
Fig. 5.1 Respiratory chain of protozoa, plants, and fungi. For explanation, see the text
Already during early studies it was shown that oxygen consumption in plant
mitochondria (in contrast to animal mitochondria) is cyanide-resistant. This
activity was later shown to be connected to the presence of a special enzyme
alternative cyanide-resistant terminal oxidase (AOX) inherent in the mitochondria
of protozoa, plants, and fungi.1 This protein consists of just one polypeptide of
molecular mass of about 40 kDa. The alternative oxidase is a peripheral membrane
protein (it has no transmembrane a-helices) and, similarly to NDin, is attached to
the inner side of the inner mitochondrial membrane (Affourtit et al. 2002). The
genes coding all the alternative enzymes of the mitochondrial respiratory chain
(AOX, NDin, and NDex) are located in the nuclear genome.
The primary sequence of AOX has certain similarities to the sequences of
different monooxygenases and, like these enzymes, the alternative oxidase contains two atoms of nonheme iron as redox cofactors. These atoms form a binuclear
center (similar to the hemecopper binuclear center of complex IV), which is
probably the site of O2 reduction. AOX is different from complex IV in a few other
important properties. For instance, it is directly ubiquinol, and not cytochrome c,
that serves as an electron donor for this enzyme (i.e., AOX is not a cytochrome
c oxidase, but a quinol oxidase). Also, alternative oxidase activity is not coupled to
transmembrane proton translocation. So, all the energy of the reaction catalyzed by
AOX is dissipated as heat (as occurs with NDin and NDex). Hence, AOX shunts the
second and third energy coupling sites of the respiratory chain.
It is important to note that the alternative oxidase activity is controlled by a
large number of cell metabolism parameters. First of all, high activity of plant
1
122
123
Fig. 5.2 Respiratory chain of the soil bacterium P. denitrificans. See explanation in the text
124
5:1
The anaerobic redox chain of P. denitrificans contains enzymes that catalyze all
the reactions of this process (Ferguson, 1994). The first stage of denitrification
(nitrate reduction to nitrite) is catalyzed by two different nitrate-reductases (Nap
and Nar, see Fig. 5.2). The Nar protein is a membrane enzyme and includes an
unusual cofactor that contains a molybdenum atom. Quinol serves as an electron
donor for Nar. This enzyme is a D
lH generator that functions as a Mitchell loop
with the stoichiometry of 1 H+/e. The second nitrate-reductase (Nap) is located in
the periplasm, and its activity is not coupled to proton potential generation. The
next three stages of denitrification are catalyzed by nitrite-reductase, NO-reductase, and N2O-reductase, respectively. Cytochrome c serves as an electron donor
for all of these enzymes. Activities of nitrite-reductase, NO-reductase, and N2Oreductase are not coupled to D
lH generation. However, due to cytochrome
c oxidation, these enzymes create a possibility for proton potential generation
under anaerobic conditions in the first two coupling sites of the respiratory chain
(i.e., by complexes I and III). So, functioning of these enzymes leads (though
indirectly) to the increased efficiency of energy use under the conditions of
anaerobic growth of P. denitrificans. The overall denitrification process serves as
an example of an anaerobic electron transfer, i.e., a bacterial respiratory chain
using other than O2 compounds as the terminal electron acceptors.
125
main properties. NDH-2 consists of one polypeptide, contains one redox cofactor
(FAD), and its enzymatic activity is not coupled to transmembrane proton
translocation.
Quinol, being reduced by NDH-1 or NDH-2, is then oxidized by one of two
alternative terminal quinol oxidasesa bo- or bd-type. The bd-type enzyme
becomes the main oxidase of the E. coli respiratory chain when the cells grow
under microaerobic conditions (i.e., under low O2 concentration), while the botype oxidase prevails under high O2 concentration. The bo-type quinol oxidase
consists of three subunits and contains three redox cofactors: copper CuB and two
hemesa low-spin heme B and a high-spin heme O. The heme O and CuB form a
binuclear center which serves (similar to other hemecopper oxidases) as a site for
oxygen binding and reduction. Although the bo-type oxidase is a quinol oxidase,
its primary sequence and especially its three-dimensional structure are very similar
to aa3-type cytochrome c oxidase. So, it is not surprising that the bo-type oxidase
functions as a proton pump and generates D
lH with stoichiometry of 2 H+/e
(Puustinen et al. 1991) (Fig. 5.4a).
The second terminal oxidase of E. coli (cytochrome bd) is a rather unusual
enzyme. This protein was found to be homologous neither to oxidases with heme
copper binuclear center (mitochondrial cytochrome c oxidases and similar bacterial
enzymes), nor cyanide-resistant oxidases with ironiron binuclear center (alternative oxidases of plant mitochondria, protozoa, fungi, bacteria, and chloroplasts). So,
the bd-type quinol oxidase is a representative of a third type of terminal oxidases.
This E. coli enzyme consists of two subunits and includes three prosthetic groups: a
low-spin heme b558 and two high-spin hemesheme b595 and a chlorin heme
d. High-spin hemes b595 and d are located very close to each other in the protein, and
126
127
128
Ascaris larvae that live under aerobic conditions (in blood) have mitochondria
with a typical respiratory chain and all the enzymes of the Krebs cycle.
(Footnote 5 continued)
them is oxidized to acetate while the other is converted to fumarate, which is further reduced to
succinate. The reducing equivalents for succinate production are passed from the former lactate
molecule to a quinone via NADH and complex I.
129
Fig. 5.6 Respiratory chain structure in the soil nitrogen-fixing bacterium A. vinelandii. See
explanation in the text
is equal to the value observed in the mitochondrial respiratory chain, namely 5 H+/
e. However, increased aeration and especially deficiency of fixed nitrogen induce
the respiratory chain enzymes that are noncoupled or have decreased efficiency of
energy conservation: NDH-2 and bd-type quinol oxidase (Poole and Hill 1997;
Bertsova et al. 2001). The overall coupling efficiency of this chain is only 1 H+/e.
Hence, induction of these enzymes causes a fivefold lowering of the efficiency of
proton potential generation by the entire respiratory chain.
It is due to such low efficiency that the activity of the electron transport
respiratory chain of A. vinelandii is not limited by the generated proton potential
when it becomes necessary to activate respiratory protection of the nitrogenase
complex. Besides that, NDH-2 and the bd-type quinol oxidase have very high
turnover rates. For instance, in the case of NDH-2 from E. coli, the specific activity
of the enzyme is at least 23 orders of magnitude higher than the activity of NDH1. Very fast turnovers of NDH-2 and bd-type quinol oxidase are most likely
connected to the lack of proton pumps activity of these enzymes. There is a price
that needs to be paid for the kinetic advantages of the respiratory chain consisting
of NDH-2 and bd-type quinol oxidase. It is not surprising that the growth yield
(biomass growth normalized by the amount of substrate consumed) is very low
under conditions of high aeration.
130
The solution to this problem was found when the pH-dependence of the redox
potential of the H2O/O2 couple was taken into account. Because hydrogen ions
participate in the formation of water from O2 O2 4e 4 H ! 2 H2 O, the
redox potential becomes more positive when the medium is acidified, which
moves the balance of the reaction of oxygen reduction to the right. This change
can be expressed quantitatively as 60 mV per pH unit. For instance, at pH = 3 the
redox potential of the H2O/O2 couple would be 820 mV ? (60 mV 9 4)
= 1060 mV. This value is 290 mV more positive than the redox potential of the
Fe2+/Fe3+ couple, which does not depend on pH because H+ ions do not participate
in the oxidation of ferrous ions. Such a potential difference is sufficient for ATP
synthesis. That is why iron bacteria live only at acidic pH values (the pH optimum
for A. ferrooxidans is between 3 and 1).
However, ATP synthesis is the only one problem that needs to be solved by
A. ferrooxidans. Another deals with the necessity to use an electron donor with
positive redox potential to provide biosyntheses with reducing equivalents. Acidithiobacillus ferrooxidans belongs to the group of chemolithotrophic bacteria. It is
capable of synthesizing all of its components from CO2, H2O, NH3, and mineral
salts. For these processes to be realized, a mechanism for reduction of NAD(P)+ by
Fe2+ is needed. This type of reduction consumes energy because the redox
potential for the NAD+/NADH couple (-320 mV) is far more negative than the
potential of the Fe2+/Fe3+ couple.
It is reverse electron transfer in the middle and initial segments of the respiratory chain that is used by A. ferrooxidans to overcome this difficulty. This
thermodynamically unfavorable electron transport from high to low redox
potential is catalyzed with the expense of energy of D
lH generated in the terminal
segment of the respiratory chain.
According to the scheme of energy supply of iron bacteria, the electrons taken
from Fe2+ ions enter the cytochrome oxidase system, which transfers electrons to
oxygen, this process being coupled to D
lH generation. The generated proton
potential is later used not only for ATP synthesis, but also for all the other types of
work inherent in bacterial membrane. The D
lH is also utilized for reversing the
CoQH2:cytochrome c-oxidoreductase and NADH:CoQ-oxidoreductase reactions,
which thus become D
lH consumers (instead of being D
lH generators, Fig. 5.7).
Experiments carried out with the two species of iron bacteria by Aleem (1966)
and in our group by Tikhonova et al. (1967) showed that Fe2+ ions reduce NAD+
due to reverse electron transfer that is supported by the energy produced in the
terminal segment of the respiratory chain. Acidithiobacillus ferrooxidans was also
shown to have huge amounts of cytochromes c and cytochrome oxidase, while the
components of the middle and the initial segments of the respiratory chain were
131
S2 O2
3 2 O2 H2 O ! 2 SO4 2 H
5:2
It has been already mentioned in the preceding chapter that NDH-1 of cyanobacteria contains
an unusual FP-fragment, and this protein is likely to function as a NADPH:plastoquinoneoxidoreductase.
132
5:3
0
For this reaction, the standard Gibbs free energy at pH = 7 (DG0 , see
Appendix 1) is equal to -216 kJ/mol.
Reaction 5.3 is accompanied by energy release, which can be stored in the form
of ATP during fermentation, usually through the mechanism of substrate phosphorylation. But the products of glucose fermentation still contain a rather large
133
5:4
5:5
5:6
5:7
5:8
134
5:9
135
5:11
It should be noted that the ATP hydrolysis energy and the energy of transmembrane sodium
potential are used at the initial stages of methanogenesis for the activation of CH3COOH and CO2
molecules, respectively. So, the oxidative part of methanogenesis on the whole does not lead to a
net energy storage.
10
F420 is a deazaflavin derivative (see Appendix 2, Fig. A2.11).
136
Let us discuss the possible mechanisms of energy storage during the reduction
of heterodisulfide by molecular hydrogen. This reaction takes two stages. At first, a
special enzyme, F420-independent hydrogenase, oxidizes H2 and transfers electrons
to the archaean lipophilic cofactor methanophenazine (MP), which is a functional
(but not structural) analog of the quinones of the bacterial and eukaryotic respiratory chains (Abken et al. 1998). This hydrogenase consists of three subunits
containing two B hemes, a FeS cluster, and a special prosthetic group composed of
a nonheme iron and a nickel atom. According to the scheme presented in Fig. 5.9,
H2 oxidation occurs at the NiFe center, then electrons are transferred via the FeS
cluster to cytochrome b where methanophenazine is reduced. The protons formed
during the oxidation of H2 are released to the outer medium; at the same time,
protons are taken from the cytoplasm during the formation of MPH2 from MP. So
the hydrogenase generates proton potential according to the Mitchell loop mechanism with stoichiometry of H+/e = 1.
Thus formed MPH2 can be later oxidized by the next enzyme of the methanogen electron transport chainheterodisulfide-reductase. This enzyme consists of
two subunits and contains two FeS clusters and two B hemes (in the case of
Methanosarcina barkeri the redox potentials of the hemes are -180 and 23 mV). Oxidation of MPH2 occurs at the cytochrome subunit of the
137
References
Abken HJ, Tietze M, Brodersen J, Bumer S, Beifuss U, Deppenmeier U (1998) Isolation and
characterization of methanophenazine and function of phenazines in membrane-bound
electron transport of Methanosarcina mazei G1. J Bacteriol 180:20272032
Affourtit C, Albury MS, Crichton PG, Moore AL (2002) Exploring the molecular nature of
alternative oxidase regulation and catalysis. FEBS Lett 510:121126
Aleem MI (1966) Generation of reducing power in chemosynthesis. 3. Energy-linked reduction of
pyridine nucleotides in Thiobacillus novellus. J Bacteriol 91:729736
Bertsova YV, Bogachev AV, Skulachev VP (2001) Noncoupled NADH: ubiquinone oxidoreductase of Azotobacter vinelandii is required for diazotrophic growth at high oxygen
concentrations. J Bacteriol 183:68696874
Bogachev AV, Murtasina RA, Skulachev VP (1996) H+/e stoichiometry for the NADH
dehydrogenase I and dimethyl sulfoxide reductase in anaerobically grown Escherichia coli
cells. J Bacteriol 178:62336237
Borisov VB, Gennis RB, Hemp J, Verkhovsky MI (2011) The cytochrome bd respiratory oxygen
reductases. Biochim Biophys Acta 1807:13981413
11
F420H2 can be formed also during the oxidation of H2 due to the activity of F420-dependant
hydrogenase.
138
Chapter 6
Bacteriorhodopsin
139
140
Bacteriorhodopsin
(crystal) packing in the purple membrane. There are no other proteins in this
membrane, which in fact represents 2-D crystals of bacteriorhodopsin.
Such processes as intermolecular transfer of reducing equivalents or hydrolysis
of covalent high-energy bonds, always involved in reactions catalyzed by other
D
lH generators, are absent from bacteriorhodopsin.
According to the current point of view, the mechanism of bacteriorhodopsin
functioning can be described in the following way. A photon absorbed by retinal
residue (a chromophore covalently bound with the aldimine bond to the e-amino
group of a lysine residue of the bacteriorhodopsin polypeptide) induces alltrans ? 13-cis isomerization of retinal (Fig. 6.3). This process is accompanied by
a large acidic pKa shift of the aldimine Schiff base nitrogen protonated in the dark. As
a result, the Schiff base loses H+, which appears to be released to the aqueous phase
outside the bacterial cell. Then, the pKa value of the Schiff base returns to its initial
level, i.e. the affinity of the base to protons is once again increased. As a result,
141
the Schiff base is reprotonated by an H+ ion coming from the inside of the cell (from
the cytoplasm). Then retinal returns to its initial conformation (a 13-cis ? alltrans transition). Thus, the light-induced turnover of bacteriorhodopsin is coupled
to the translocation of H+ across the cytoplasmic membrane of H. salinarium.
142
Bacteriorhodopsin
hydrophobic segments are absent from the model shown in Fig. 6.6b, since the
resolution of the method was too low. The N- and C-ends of the bacteriorhodopsin
polypeptide chain are located on the opposite sides of the cytoplasmic membrane:
the N-end is outward directed and C-end inward.
The 3-D structure of bacteriorhodopsin was later determined also by the X-ray
analysis method (*1.4 resolution) (Pebay-Peyroula et al. 1997; Schobert et al.
2002). This more detailed model of the protein structure made it possible to
identify some important elements that indicate the possible mechanism of its
functioning (Lanyi 2004). The retinal moiety was found to be connected to
Lys-216 located in the G a-helical column of the bacteriorhodopsin molecule. The
Schiff base is located in the middle of the core hydrophobic part of the protein
(Fig. 6.7). Two aspartic acid residuesAsp-85 and Asp-212are located next to
the Schiff base. The Asp-85 residue is connected to the outer medium via a protontransferring pathway that consists of the Arg-82, Glu-194, and Glu-204 residues,
143
and also of seven or eight firmly bound water molecules. Thus, the Schiff base
deprotonation in the bacteriorhodopsin photocycle can lead to export of the
released proton to the extracellular medium. In contrast, only one polar amino acid
residue (Asp-96) is located between the Schiff base and cytoplasmic membrane
surface in bacteriorhodopsin. The distances between the cytoplasmic membrane
surface and Asp-96 and further between Asp-96 and the Schiff base (10-12 ) are
too far for the direct proton transfer. Hence substantial conformational changes
should occur in order for the Schiff base to be reprotonated by cytoplasmic H+
ions. These changes really take place. They result in the formation of a very
narrow water-filled cleft leading from the cytoplasm to Asp-96 and further to the
Schiff base. Only about 10 H2O molecules can find space in this cleft. Certain
structural properties of bacteriorhodopsin indicate the possible mechanism of such
conformational changes. The B, C, and F columns are bent due to the presence of
proline residues in these a-helices. Column G also has a mobile structural element.
The a-helix in this segment is interrupted by one turn of p-helix (p-bulge). This
element is formed due to the fact that oxygen carbonyl atoms in the peptide group
of Ala-215 and Lys-216 form hydrogen bonds not with the hydrogen atoms of the
144
Bacteriorhodopsin
following peptide groups (as in a-helices), but with water molecules. Thus, a
p $ a transition is possible in this place, a feature facilitating light-induced
structural changes in the cytoplasmic part of bacteriorhodopsin, resulting in reprotonation of the Schiff base by cytoplasmic H+ ions (Lanyi 2004).
145
146
Bacteriorhodopsin
147
148
Bacteriorhodopsin
binding center of the protein. This is why the conformation of retinal in K590
intermediate was called twisted. Thus, the energy of the absorbed photon is
stored in the form of a strained conformation of the retinal moiety at the first stage
of the photocycle.1
2. K590 ? L550 transition
During the next stage, retinal residue changes from the strained conformation to
the relaxed 13-cis conformation. In fact, twisted retinal residue pushes apart
amino acid residues of the protein which acquires a new strained conformation
with a changed retinal-binding site (at this stage, energy is transferred from retinal
to the protein).
3. L550 ? MI412 transition
The Schiff base and Asp-85, when in the new protein conformation, change
their affinities to H+ so that their pKa shift to approximately 8 (the reasons for these
changes are still unknown). This event initiates proton transfer from the Schiff
base to Asp-85.
4. MI412 ? MII412 transition
Deprotonation of the Glu-204 residue on the outer bacteriorhodopsin surface
occurs at this stage. As a result, a proton is released to the outer medium.
5. MII412 ? MIII
405 transition
At this stage, substantial conformational changes occur in the bacteriorhodopsin
molecule. The structure of the mobile elements of the a-helices is probably
changed (see above) and a cleft is formed between a-columns in the cytoplasmic
part of bacteriorhodopsin. It is due to these changes that water penetrates the space
between Asp-96 and the Schiff base, facilitating proton transfer between these two
groups.
6. MIII
405 ? N560 transition
The change in the surroundings of the Asp-96 residue shifts its pKa to *7. This
in turn results in proton transfer from Asp-96 to the Schiff base. Thus, reprotonation of the bacteriorhodopsin prosthetic group occurs at this stage.
7. N560 ? O640 transition
Reverse isomerization of 13-cis retinal to the energetically more favorable alltrans state occurs at this stage. Also, the Asp-96 residue is reprotonated due to the
proton uptake from the cytoplasm, and the cleft in the cytoplasmic part of the
bacteriorhodopsin molecule closes.
Two intermediates in the bacteriorhodopsin photocycle before K590 were discovered using
spectroscopy with sub-picosecond resolutiona truly initial intermediate I480, and J625 which
is formed from I480 within 700 fs (710-13 s).
149
150
Bacteriorhodopsin
151
152
Bacteriorhodopsin
153
154
Bacteriorhodopsin
Animal rhodopsin converted to MII acquires the ability for specific interaction
with another protein called transducin. This interaction induces replacement of
GDP by GTP in transducin. Such an event initiates the following processes.
A complex of transducin and GTP activates a cGMP-specific phosphodiesterase,
causing, as a result, a decrease in the cGMP concentration in the photoreceptor
cell. The latter event entails closing Na+ channels in the outer membrane of these
cells. The channels in question were found to require cGMP to be open. Closing of
the channels lowers the membrane conductance and therefore increases DW across
the plasma membrane (this DW is continuously generated by the Na+/K+-ATPase
of the plasma membrane of the photoreceptor cells). A DW increase means
excitation of the photoreceptor cell, which is recognized by special neurons
transmitting the light signal to a corresponding center in the brain.
All these processes are assumed to participate in amplification of the light
signal. For instance, one molecule of the long-lived intermediate MII facilitates
GDPGTP exchange in hundreds of transducin molecules. Moreover, one phosphodiesterase activated by the GTPtransducin complex hydrolyzes hundreds of
molecules of cGMP before the active phosphodiesterasetransducinGTP complex
decomposes due to the hydrolysis of the bound GTP to GDP and Pi. Taking these
facts into account, one can understand how the rod cell can be excited by such a
small amount of light energy as a single photon.
Amplification, however, does not appear to be necessary and is even undesirable at high light intensities when, for example, a million photons, rather than a
single photon should be sensed. In this case, a linear rather than exponential
relationship between the light intensity and its effect seems to be required. It has to
be noted that the electric potential difference generated by rhodopsin in the cone
cells plasma membrane is of the same direction as the one generated by Na+/K+ATPase (the cell interior is negative). This means that the ability of rhodopsin for
light-induced DW generation may be used for perception of a strong and sudden
increase in illumination, and this response will be much faster than in the case of a
complicated system of light signal amplification used in rod cells in low light
intensity. Another interesting feature is that the response to intense light will
spontaneously disappear over time, as rhodopsin is a photoelectric generator of a
single action: photon absorption leads to the splitting of the retinal moiety from
rhodopsin, and in order for the initial protein form to be regenerated, 11-cis retinal
needs to be once again attached to aporhodopsin. It is noteworthy that in cones and
rods, rhodopsin is localized in the outer cell membrane and photoreceptor disks,
respectively. The latter are not connected to the outer cell membrane; the rhodopsin-generated DW increase can directly excite cones but not rods, the fact
consistent with well-known functional difference between cones responding to
bright light, and rods responding to dim light. It should be stressed that cones,
besides direct rhodopsin-mediated excitation in response to strong and sudden
increase in a light intensity, are equipped, like rods, by a mechanism of signal
amplification which may be used for perception of steady changes in low light
intensities (Skulachev 1988).
References
155
References
Balashov SP, Lanyi JK (2007) Xanthorhodopsin: proton pump with a carotenoid antenna. Cell
Mol Life Sci 64:23232328
Baryshev VA, Glagolev AN, Skulachev VP (1981) Sensing of D
lH in phototaxis of
Halobacterium halobium. Nature 292:338340
Bj O, Aravind L, Koonin EV, Suzuki MT, Hadd A, Nguyen LP, Jovanovich SB, Gates CM,
Feldman RA, Spudich JL, Spudich EN, DeLong EF (2000) Bacterial rhodopsin: evidence for
a new type of phototrophy in the sea. Science 289:19021906
Bj O, Spudich EN, Spudich JL, Leclerc M, DeLong EF (2001) Proteorhodopsin phototrophy in
the ocean. Nature 411:786789
Belyakova TN, Kadzyaukas YuP, Skulachev VP, Smirnova IA, Chekulaeva LN, Jasaitis AA
(1975) Generation of electrochemical potential of H+ ions and photophosphorylation in the
cells of Halobacterium halobium. Dokl Akad Nauk SSSR 223:483486
Bibikov SI, Grishanin RN, Kaulen AD, Marwan W, Oesterhelt D, Skulachev VP (1993)
Bacteriorhodopsin is involved in halobacterial photoreception. Proc Natl Acad Sci U S A
90:94469450
Bogomolni RA, Spudich JL (1982) Identification of a third rhodopsin-like pigment in phototactic
Halobacterium halobium. Proc Natl Acad Sci U S A 79:62506254
Drachev LA, Jasaitis AA, Kaulen AD, Kondrashin AA, Liberman EA, Nemecek IB, Ostroumov
SA, Semenov AYu, Skulachev VP (1974) Direct measurement of electric current generation
by cytochrome oxidase, H+-ATPase and bacteriorhodopsin. Nature 249:321324
Drachev LA, Kaulen AD, Skulachev VP (1984) Correlation of photochemical cycle, H+ release
and uptake, and electric events in bacteriorhodopsin. FEBS Lett 178:331335
Drachev LA, Kaulen AD, Skulachev VP, Zorina VV (1986) Protonation of a novel intermediate P
is involved in the M-bR step of the bacteriorhodopsin photocycle. FEBS Lett 209:316320
Gerber GE, Anderegg RJ, Herlihy WC, Gray CP, Biemann K, Khorana HG (1979) Partial
primary structure of bacteriorhodopsin: sequencing methods for membrane proteins. Proc Natl
Acad Sci U S A 76:227231
Grigorieff N, Ceska TA, Downing KH, Baldwin JM, Henderson R (1996) Electron-crystallographic refinement of the structure of bacteriorhodopsin. J Mol Biol 259:393421
Harbison GS, Smith SO, Pardoen JA, Winkel C, Lugtenburg J, Herzfeld J, Mathies R, Griffin RG
(1984) Dark-adapted bacteriorhodopsin contains 13-cis, 15-syn and all-trans, 15-anti retinal
Schiff bases. Proc Natl Acad Sci U S A 81:17061709
Henderson R, Unwin PN (1975) Three-dimensional model of purple membrane obtained by
electron microscopy. Nature 257:2832
Kalaidzidis IV, Kaulen AD, Radionov AN, Khitrina LV (2001) Photoelectrochemical cycle of
bacteriorhodopsin. Biochemistry (Mosc) 66:12201233
Kolbe M, Besir H, Essen LO, Oesterhelt D (2000) Structure of the light-driven chloride pump
halorhodopsin at 1.8 resolution. Science 288:13901396
Lanyi JK (2001) X-ray crystallography of bacteriorhodopsin and its photointermediates: insights
into the mechanism of proton transport. Biochemistry (Mosc) 66:11921196
Lanyi JK (2004) Bacteriorhodopsin. Annu Rev Physiol 66:665688
Lozier RH, Bogomolni RA, Stoeckenius W (1975) Bacteriorhodopsin: a light-driven proton
pump in Halobacterium halobium. Biophyl J 15:955962
Lozier RH, Niederberger W, Bogomolni RA, Hwang S, Stoeckenius W (1976) Kinetics and
stoichiometry of light-induced proton release and uptake from purple membrane fragments,
Halobacterium halobium cell envelopes, and phospholipid vesicles containing oriented purple
membrane. Biochim Biophys Acta 440:545556
Mukohata Y, Kaji Y (1981) Light-induced membrane-potential increase, ATP synthesis, and
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N-dicyclohexylcarbodiimide, triphenyltin chloride, and 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847). Arch Biochem Biophys 206:7276
156
Bacteriorhodopsin
Part III
DlH Consumers
Chapter 7
D
lH -Driven Chemical Work
159
7 D
lH -Driven Chemical Work
160
Fo
a
b
c
d
e
a
b
c
a
b
c
OSCP
d and e
a
b
c
55.0
50.0
31.5
19.5
15.0
30.0
17.0
8.0
513
459
287
177
138
271
156
79
Number of
subunits per FoF1
3
3
1
1
1
1
2
10
living organisms (see previous chapters). H+-ATP synthase can be easily dissociated into two subcomplexes, one responsible for transmembrane proton transport
(Fo) and the other for ATP synthesis or hydrolysis (F1).1
For the E. coli H+-ATP synthase, the subunit composition, amino acid
sequence, and operon structure have been established (Bragg 1984). It was found
that the E. coli factors F1 and Fo are composed of five (named alphabetically from
a to e) and three (a, b, and c) types of subunits, respectively (see Table 7.1). The
stoichiometry of subunits was shown to be 3a:3b:c:d:e:a:2b:10c.2 The molecular
masses of F1, Fo and the total H+-ATP synthase complex from E. coli are 381,
*145, and *525 kDa, respectively.
The FoF1 complex of E. coli is encoded by a single operon called unc or atp. It
is about seven thousand base pairs in size. The order of the genes corresponds to
the following order of transcription of mRNA coding, i.e., I, a, b, c, d, a, c, b, and e
subunits where I is a hypothetical protein with unknown functions that is absent
from purified FoF1.
Mitochondrial H+-ATP synthase is also composed of subcomplex F1 (six types
of main subunits), subcomplex Fo (three types of main subunits), and several
additional subunits (Walker et al. 1985). The F1 factor contains 3a, 3b, c, d, and e
subunits and the oligomycin sensitivity conferring protein (OSCP) protein. The
major subunits (a and b) resemble very much their E. coli analogs. For instance, b
subunits of F1 factor from bovine and yeast mitochondria, from E. coli, and from
chloroplasts have about 70 % conservative sequences. This is also true for the a
subunit.
A comparison of minor F1 subunits from E. coli and animal mitochondria
showed that the d and e subunits are different, while the c subunits are similar
(Walker et al. 1982) (see also Sect. 7.1.2). In the first approximation, we can say
that mitochondrial subunits d and e correspond to the e subunit of E. coli. Mitochondrial factor F1 was shown to contain an additional polypeptide, the so-called
1
Besides ATP synthases of FoF1-type, ATP synthases (ATPases) of V0V1- and E1E2-types may
be also found in living organisms. The last sections of this chapter deal with these enzymes.
2
The number of c-type subunits of Fo factor will be discussed in more detail later.
161
OSCP. This protein has many homologies with the E. coli d subunit, but some
differences between these two proteins were also found. OSCP is slightly larger
(21 kDa instead of 19.3 kDa of the E. coli d subunit); it has a hydrophobic segment close to the N-terminus, and there are several pieces in the sequence
homologous not to the d, but to the b subunit of the E. coli enzyme.
The structure of the Fo factor from animal mitochondria is similar to that from
E. coli. Polypeptides a, b, and c are its main subunits. Subunit b from animal
mitochondria was shown to be slightly different from the bacterial analog, and
animal Fo factor probably contains only one b-type polypeptide (instead of two in
bacteria). The following stoichiometry of the main subunits of the animal ATP
synthase is now generally accepted: 3a:3b:c:d:e:OSCP:a:b:8c.
Several additional polypeptides in mitochondrial H+-ATP synthase are absent
from the bacterial enzyme. These are: A6L subunit of Fo factor; factor F6 (9 kDa);
9.5 kDa protein inhibitor of F1 factor; 18.5 kDa d subunit (which, together with F6
and OSCP is required for correct binding of F1 and Fo); and several other subunits.
In animals and yeasts, all the F1 subunits are encoded in the nuclear genome
and synthesized in the cytoplasm. Among Fo components of the yeast enzyme,
subunits a, c, and A6L are encoded by the mitochondrial genome; all the others are
encoded by the nuclear genome. In DNA of animal mitochondria, there are two
overlapping genes encoding subunits a and A6L of Fo; all the other subunits
(including c) are encoded in the nucleus.
The subunit composition of the chloroplast H+-ATP synthase (it is usually
referred to as CFoCF1) is, in principle, similar to that of bacteria and mitochondria
(Richter et al. 2000). Again, factor F1 has the 3a:3b:c:d:e structure. The e subunit
is homologous to the bacterial one. However, the CFo factor contains not three, but
four types of main subunitsa, b, b0 , and c (subunits b and b0 are homologous)
with the suggested stoichiometry of a:b:b0 :14c. In other words, CFo contains not a
homodimer of b subunits (as in bacteria), but a heterodimer of two different
(though rather similar) subunits b and b0 .
162
7 D
lH -Driven Chemical Work
spherical particles with the membrane vesicles was shown to restore ATP-synthase
and ATPase activity in the latter. From these observations, Racker concluded that
the knobs represent the catalytic part of the H+-ATP synthase, i.e., factor F1. A part
of H+-ATP synthase responsible for H+-ion transfer (factor Fo), was shown to
remain in the membrane after the detachment of factor F1. This fact led to a substantial increase in proton conductivity of the membrane. The results of a computer
analysis of electron micrographs of factor F1 are summarized in Fig. 7.2. The
diameter of the frontal projection of factor F1 is about 10 nm.
The 3D structure of the main part of mitochondrial factor F1 has been established with the high resolution of 2.4 by X-ray analysis (Abrahams et al. 1994).
163
This work was recognized by the Nobel Prize in Chemistry in 1997 to John Walker
(Fig. 7.3). As shown in Fig. 7.4, the greater part of F1 is composed of subunits a,
b, and c with stoichiometry of 3:3:1. The a and b subunits form a spherical globule
(Stock et al. 2000). The central part of this globule consists of very long a-helical
Fig. 7.4 Structure of the
main part of factor F1 from
mitochondrial H+-ATP
synthase. Different subunits
are marked with the
following colors: ared,
byellow, cblue,
dgreen, epurple (Stock
et al. 2000)
164
7 D
lH -Driven Chemical Work
parts of the c subunit; subunits a and b are located alternately around the c subunit
(this structure is often compared to that of an orange). It seems important to note
that the c subunit occupies an asymmetrical position in the center of the globule.
Subunit c protrudes significantly beyond the 3a:3b-globule on the one of its sides.
This protruding part of subunit c together with subunits d and e form the central
stalk of factor F1 that connects it to factor Fo.
Subunits a and b contain one nucleotide-binding center each. A nucleotidebinding domain has also been identified in c subunit. However, under physiological conditions this domain is most likely not able to bind nucleotides.
Only indirect information is available concerning the 3D structure of the rest of
ATP synthase. These data were obtained using such methods as electron diffraction, NMR, and X-ray diffraction analysis of isolated subunits, and different
possibilities of their cross-linking in FoF1 complex. A scheme of the FoF1 complex
structure is presented at Fig. 7.5.
The 3D structure of the c subunit from the E. coli enzyme in a hydrophobic
solvent was determined by NMR. This subunit was shown to consist of two
transmembrane a-helical columns that are connected to each other on the cytoplasmic membrane side by a loop of polar amino acid residues (Fig. 7.6). The
C-terminal a-helical column contains a conservative aspartate residue (Asp-61)
that plays a key role in transmembrane proton transport. This residue seems to be
located approximately in the middle of the hydrophobic layer of the membrane. In
factor Fo the c-type subunits form an oligomer consisting of 814 c-type subunits.
Recently, the structure of this c oligomer of Na+-ATP synthase from the bacterium
Ilyobacter tartaricus has been determined by X-ray diffraction (Meier et al. 2005).
The a-helical columns of c subunits form in the membrane a kind of repeating
Fig. 7.5 Scheme illustrating current understanding of the structure of FoF1-ATP synthase from
eukaryotic mitochondria (left) and bacteria (right) (Stock et al. 2000). The two b subunits
(yellow) are basically covered by a subunits; the third b subunit is not shown. The A6L subunit as
well as other small subunits of the mitochondrial factor Fo absent from the bacterial subcomplex
are also not shown
165
system consisting of two rings, one being inside the other (Fig. 7.7). The external
and internal rings consist of C-terminal and N-terminal a-helical columns,
respectively. The number of c subunits is supposed to vary in ATP synthases of
different organisms. For instance, ATP synthase from animal mitochondria is
considered to contain 8 c subunits; the yeast mitochondria enzyme 10 c subunits;
the chloroplast ATP synthase 14 c subunits; and the sodium ATP synthase from
I. tartaricus 11 c subunits (Stock et al. 2000). We will discuss later that the
variable stoichiometry of c subunits can play an important physiological role for it
leads to a variable ratio of H+/ATP.
The oligomer of c subunits is connected to factor F1 through the central stalk of
the FoF1 complex (Fig. 7.8). It is important to note that the central stalk is located
Fig. 7.7 Structure of the c ring of Na+-ATP synthase from I. tartaricus: a view perpendicular to
the membrane plane, b view parallel to the membrane plane. Individual c subunits are marked
with different colors. Blue spheres represent bound sodium ions (Meier et al. 2005)
166
7 D
lH -Driven Chemical Work
Fig. 7.8 Stereo image of the F1-c complex from S. cerevisiae mitochondria. Side view (a) and
bottom view (b). Schemes showing the arrangement of subunits are presented in the lower right
corners. Peripheral stalk is not seen (Stock et al. 2000)
167
It should be stressed that in reality all the nucleotide-binding sites are located in the area of a
and b subunit contacts. But in order to simplify the description, we will attribute these sites to
those subunits which possess more amino acid residues participating in the formation of the
corresponding sites.
168
7 D
lH -Driven Chemical Work
169
170
7 D
lH -Driven Chemical Work
Fig. 7.11 Binding of ADP and Pi in the active site of factor F1. The numbers near the dotted
lines represent the distance in angstroms
Fig. 7.12 Nobel Laureate
Paul D. Boyer
Fo modifier, DCCD or by a specific FoF1ATPase inhibitor, oligomycin. Reconstitution of the FoF1 complex suppresses the leakage as well (Friedl et al. 1983).
In bacteria, it has been shown that H+ conductance does not occur in F1-stripped
membranes from mutant strains in which any of the three Fo subunits has been lost. It
seems very probable that it is the a and c subunits that play the leading role in proton
transport, and the transmembrane proton transport pathway is formed at the site of
contact of these subunits. There is an aspartate or glutamate residue in the c subunits
171
of Fo from different organisms that is absolutely necessary for protons transfer (in E.
coli it is Asp-61). It is located about 1.8 nm from the membrane surface, i.e., in the
middle of the hydrophobic core of the membrane. Its replacement with Asn or Gln
completely abolishes both the H+ transport and D
lH -powered ATP synthesis
(Hoppe et al. 1982). Transmembrane a-helical columns of the a subunit also contain
several conservative charged amino acid residues (Arg-210, His-245, Glu-196, and
Glu-219). These residues probably participate in the formation of the proton
transport pathway.
Sensitivity of Fo to DCCD is a characteristic feature of this protein. DCCD is a
covalent modifier of protonated carboxylic groups. At low concentrations, it binds
selectively to Asp-61 of the c subunits of ATP synthase (Negrin et al. 1980). This
modification of Asp-61 completely abolishes the H+-conducting function of Fo. It
seems worth noting that modification of only one c subunit in Fo seems to be
sufficient for complete inactivation of the enzyme.
Oligomycin was shown to be another effective inhibitor of mitochondrial factor
Fo (besides the already mentioned DCCD). Yeast mutants were obtained with H+conductance through factor Fo that are resistant to this antibiotic. Two loci of the
a subunit were shown to be responsible for this resistance. Both were shown to be
conservative in humans and mice. However, only one of the conservative loci was
found in the a subunit of E. coli, a fact that is in accordance with data on the weak
effect of oligomycin on the ATP synthase of this bacterium.
Factor Fo is probably not a simple pore because the rate of H+ translocation
through Fo obeys saturation kinetics with optimum at pH 79. These data indicate
the presence of several H+ binding sites. Factor Fo probably has two H+-binding
sites located on the two sides of the membrane. The highest values of H+ conductance were obtained for factor Fo from chloroplasts and photosynthesizing
bacteria: *6,000 H+ per second per Fo at DW = 100 mV (Junge 1987). However,
even this value seems to be too small for a channel-type H+-conducting
mechanism.
A functional model of factor Fo was suggested (Junge et al. 2001) that explains
all the mentioned structural features of Fo, kinetic characteristics of proton
transport by this factor, and also its DCCD-induced inactivation (Fig. 7.13). This
model is based on the assumption of two proton half-channels (located on the
opposite membrane sides) being present in factor Fo. Proton transfer between these
Fig. 7.13 Tentative scheme
of the mechanism of proton
transport by factor Fo.
Subunits a and c are colored
green and yellow,
respectively (Junge et al.
2001)
172
7 D
lH -Driven Chemical Work
two half-channels proceeds parallel to the membrane surface due to the transfer of
the protonated aspartate (glutamate) residue of a c subunit. Such movement of a
carboxylic group can be achieved due to rotation of the c ring relative to the
a subunit. During this process, the given carboxylic group has to be deprotonated
when interacting with the a subunit, and it has to be protonated when leaving this
contact. Hence, proton transport via factor Fo has to be coupled to c ring rotation.
173
Fig. 7.14 Scheme of experiment visualizing rotation of factor F1 c subunit during hydrolysis of
ATP. Factor F1 subunits are colored as follows: apink; borange; cgreen (Noji et al. 1997)
174
7 D
lH -Driven Chemical Work
of factor Fo together with the subunits c and e4 (i.e., with the central stalk of factor
F1) form the rotor of this motor, while all the other subunits of H+-ATP synthase
serve as the stator (see above, Fig. 7.5). The role of the peripheral stalk that also
connects factors Fo and F1 becomes clear within this hypothesis. This additional
connection of the two factors is probably needed for the attachment of the 3a:3b
globule to the membrane part of the enzyme stator, preventing the rotation of the
3a:3b globule together with the central stalk (Stock et al. 2000).
It should be stressed that many questions concerning the functioning of the
FoF1-ATP synthase remain unclear. First of all, we do not understand the mechanism of the coupling of proton transport in factor F1 to the rotation of the c ring.
Electrostatic interaction of the deprotonated anion Asp-61 on the c subunit (rotor)
and the cation of one of the positively charged amino acid residues of the a subunit
(stator) is suggested to play the key role in this process.
This is true for the bacterial type enzyme; in the case of the mitochondrial enzyme, the central
stalk is composed of subunits c, d, and e.
175
These data are in accordance with the experimentally measured H+/ATP stoichiometry for
chloroplast H+-ATP synthase. In the case of plants, ADP is photophosphorylated on the external
side of the thylakoid membrane. The ATP obtained is mainly utilized in the chloroplast stroma
176
7 D
lH -Driven Chemical Work
(Footnote 5 continued)
during the synthesis of glucose. No porters participate in this process, and the H+/ATP ratio was
shown to be about four protons per ATP molecule.
lH Generators
7.2 H+-ATPases as Secondary D
177
category of H+-ATPases (see Table 7.2). Let us consider these types of enzymes in
more detail.
H+-ATPase
H+/K+-ATPase
H+-ATPase
H+-ATPase
E1E2
FoF1
FoF1
V0V1
V0V1
E1E2
H+-ATPase
H+-ATPase
Mechanism
178
7 D
lH -Driven Chemical Work
lH Generators
7.2 H+-ATPases as Secondary D
179
6
7
180
7 D
lH -Driven Chemical Work
lH Generators
7.2 H+-ATPases as Secondary D
181
E1E2-type of ATPases forming a vanadate- and hydroxylamine-sensitive aspartylphosphate intermediate when ATP is hydrolyzed. Diethylstilbestrol and DCCD are
also inhibitory. This H+-ATPase is a single polypeptide with a molecular mass
slightly above 100 kDa. Its content in the plasmalemma reaches 15 % of the total
protein. The mechanism of ATP hydrolysis is shown in Fig. 7.18. H+-ATPases
from plant and fungi outer membranes have been reconstituted into proteoliposomes, and the reversibility of ATP hydrolysis was demonstrated in this system
(DW artificially created by valinomycin-mediated K+ diffusion was used for ATP
synthesis in these experiments).
There must be some physiological significance to the fact that the pH optimum
of plasmalemma H+-ATPase is between 6.0 and 7.0. At pH above 7.0, the activity
of the enzyme decreases drastically. The impression is that the enzyme is used to
prevent acidification of the cytoplasm by removing excess H+ ions when the
intracellular pH drops below neutrality. Apparently, intracellular pH regulation is
a function of the plasmalemma H+-ATPase. However, it is obvious that this
ATPase also performs another function, namely, formation of D
lH , which is then
utilized for the uphill import of metabolites into the cell that are transported via
H+,metabolite-symporters (see Chap. 9).
7.2.3.2 H+/K+-ATPase of Gastric Mucosa
A unique D
lH -generating ATPase was discovered in the gastric mucosa outer
cell membrane (Ganser and Forte 1973). It catalyzes an electroneutral exchange of
intracellular H+ for extracellular K+, in this way acidifying the gastric juice. The
enzyme is called H+/K+-ATPase. It has been purified and reconstructed into
proteoliposomes.
H+/K+-ATPase was shown to belong to E1E2 type; the enzyme contains three
polypeptides of molecular mass of about 100 kDa (Shin et al. 1997). A phosphorylated intermediate is formed in the course of the hydrolysis reaction. The
enzyme activity is inhibited by vanadate and hydroxylamine. A peculiar feature of
182
7 D
lH -Driven Chemical Work
183
7:1
7:2
protein synthesis:
amino acid tRNA ATP ! aminoacyl-tRNA AMP PPi
7:3
7 D
lH -Driven Chemical Work
184
7:4
7:6
185
7 D
lH -Driven Chemical Work
186
7.4 H+-Transhydrogenase
NAD(H) and NADP(H) are the main reducing equivalent carriers in the cell
cytoplasm of the majority of living organisms. The only structural difference
between these two cofactors is one phosphate residue. However, this minor difference appears to be sufficient to metabolically isolate NAD(H) and NADP(H)
from each other. NAD+ is practically always used for substrate oxidation in catabolic reactions, while NADPH is mainly utilized in reductive processes involved
in anabolic reactions. That is why NADP(H) is usually reduced in a cell
([NADP+] [NADPH]), while NAD(H) is mainly present in its oxidized form
([NAD+] C [NADH]).
Such reactions as glucose oxidation in the pentose phosphate pathway or isocitrate oxidation by NADP+-dependant isocitrate dehydrogenase in the Krebs cycle
are important mechanisms of NADP+ reduction to NADPH. Besides these reactions, there is also a possibility of direct hydride ion (H-) transfer from NADH to
NADP+ (Eq. 7.1), which is catalyzed by enzymes called transhydrogenases:
NADH NADP $ NAD NADPH
7:7
7.4 H+-Transhydrogenase
187
This activity was first described in 1952 by Colowick et al. (1952). Because
NADP(H) is mainly present in its reduced state while NAD(H) is oxidized, NADP+
reduction at the expense of NADH oxidation is possible only when coupled to
utilization of an additional energy source. In 1963, Danielson and Ernster (Fig. 7.21)
found that in inside-out submitochondrial particles energization strongly shifts the
equilibrium of reaction 7.1 to the right, i.e., in the direction of NADPH formation
(Danielson and Ernster 1963). As a result, the [NADPH] 9 [NAD+]/
[NADP+] 9 [NADH] ratio reached 500, whereas in nonenergized vesicles it was
close to 1, consistent with the almost equal standard redox potentials (E00 ) of the
NADH/NAD+ and NADPH/NADP+ couples.
In 1966, Mitchell proposed that transhydrogenase is an additional energycoupling site in the respiratory chain. It was suggested that oxidation of NADPH
by NAD+ (the forward reaction) generated D
lH of the same direction as respiration, whereas the reverse process consumed respiration-produced D
lH
(Mitchell 1966).
Experiments conducted by Efim Liberman et al. and the group of one of the
authors (V.P.S.) confirmed this hypothesis (Dontsov et al. 1972). Inside-out submitochondrial vesicles, Rh. rubrum chromatophores, and proteoliposomes containing purified transhydrogenase were shown to generate DW (+ inside the
vesicle) when oxidizing NADPH by NAD+. NADH oxidation by NADP+ also
caused DW formation but of the opposite direction (- inside the vesicle). Both
the first and the second types of electrogenesis were shown to be dissipated when
the concentrations of the substrates and products of this reaction were equalized. In
both cases, the generated DW was a linear function of the ratio
ln([NADPH] 9 [NAD+]/[NADP+] 9 [NADH]).
7 D
lH -Driven Chemical Work
188
NADP NADH n H
in $ NADPH NAD n Hout :
7:8
S
P
7:9
where DG0 is the energy change when [S] = [P]. In all the D
lH generators other
than transhydrogenase, DG0 is rather large (usually about 3045 kJmol-1). In the
transhydrogenase reaction, however, DG0 is close to zero. Therefore, this enzyme
can effectively compete with respiration or ATPase in D
lH formation only if
[NADPH] 9 [NAD+] are several orders of magnitude higher than
[NADP+] 9 [NADH], a most unlikely situation for the living cell.
Membrane-bound H+-transhydrogenase has been found in the inner mitochondrial membrane of many eukaryotes (first of all in higher animals), and also in
cytoplasmic membrane of the majority of bacteria.8 The mitochondrial enzyme
consists of just one polypeptide of *120 kDa molecular mass, but in vivo the
transhydrogenase functions as a homodimer (Jackson 2003). There are three
domains in the proteindI, dII, and dIII. The dI fragment is a typical nucleotidebinding domain (of the Rossmann fold type) that binds specifically NAD+ and
NADH. This specificity is reached due to hydrogen bond formation between the
conservative aspartate residue of the dI domain and the 20 -OH group of the
NAD(H) ribose residue. The dIII fragment is also a nucleotide-binding domain of
Rossmann type, but this fragment is capable of binding only NADP+ and NADPH.
In this case, specificity is reached due to the interaction of conservative lysine,
arginine, and serine residues of the dIII domain with the phosphate residue in the
NADP(H) molecule. The dII fragment is a very hydrophobic polypeptide. It is
practically completely immersed in the membrane. It is composed of 1214
(depending on the object) transmembrane a-helical columns connected to each
other by very short hydrophilic loops. The dI and dIII domains are supposed to
catalyze hydride ion transfer between NADH and NADP+, while the dII fragment
Besides the membrane-bound transhydrogenase, the cells may also have a soluble transhydrogenase that catalyzes hydride ion transfer from NADPH to NAD+ in an energy-independent
fashion. We will not discuss this enzyme in this book.
7.4 H+-Transhydrogenase
189
190
7 D
lH -Driven Chemical Work
191
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F1-ATPase from bovine heart mitochondria. Nature 370:621628
Baltscheffsky H, Von Stedingk LV, Heldt HW, Klingenberg M (1966) Inorganic pyrophosphate:
formation in bacterial photophosphorylation. Science 153:11201122
Baltscheffsky M (1969) Energy conversion-linked changes of carotenoid absorbance in
Rhodospirillum rubrum chromatophores. Arch Biochem Biophys 130:646652
Baltscheffsky M, Schultz A, Baltscheffsky H (1999) H+-PPases: a tightly membrane-bound
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Baykov AA, Dubnova EB, Bakuleva NP, Evtushenko OA, Zhen RG, Rea PA (1993) Differential
sensitivity of membrane-associated pyrophosphatases to inhibition by diphosphonates and
fluoride delineates two classes of enzyme. FEBS Lett 327:199202
Belitser VA, Tsibakova ET (1939) The mechanism of phosphorylation associated with
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Bizouarn T, Fjellstrm O, Meuller J, Axelsson M, Bergkvist A, Johansson C, Gran Karlsson B,
Rydstrm J (2000) Proton translocating nicotinamide nucleotide transhydrogenase from E.
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Boekema EJ, Berden JA, van Heel MG (1986) Structure of mitochondrial F1-ATPase studied by
electron microscopy and image processing. Biochim Biophys Acta 851:353360
Boyer PD (2002) Catalytic site occupancy during ATP synthase catalysis. FEBS Lett 512:2932
Boyer PD, Cross RL, Momsen W (1973) A new concept for energy coupling in oxidative
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Natl Acad Sci U S A 70:28372839
Bragg PD (1984) The ATPase complex of Escherichia coli. Can J Biochem Cell Biol 62:
11901197
Chow DC, Forte JG (1995) Functional significance of the beta-subunit for heterodimeric P-type
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Colowick SP, Kaplan NO, Neufeld EF, Ciotti MM (1952) Pyridine nucleotide transhydrogenase.
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Danielson L, Ernster L (1963) Energy-dependent reduction of triphosphopyridine nucleotide by
reduced diphosphopyridine nucleotide, coupled to the energy-transfer sytem of the respiratory
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Dontsov AE, Grinius LL, Jasaitis AA, Severina II, Skulachev VP (1972) A study on the
mechanism of energy coupling in the redox chain. I. Transhydrogenase: the fourth site of the
redox chain energy coupling. J Bioenerg 3:277303
Feldman RI, Sigman DS (1982) The synthesis of enzyme-bound ATP by soluble chloroplast
coupling factor 1. J Biol Chem 257:16761683
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lH -Driven Chemical Work
Frangione B, Rosenwasser E, Penefsky HS, Pullman ME (1981) Amino acid sequence of the
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Friedl P, Hoppe J, Gunsalus RP, Michelsen O, von Meyenburg K, Schairer HU (1983) Membrane
integration and function of the three Fo subunits of the ATP synthase of Escherichia coli K12.
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Ganser AL, Forte JG (1973) K+-stimulated ATPase in purified microsomes of bullfrog oxyntic
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Grubmeyer C, Penefsky HS (1981) Cooperatively between catalytic sites in the mechanism of
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Hoppe J, Schairer HU, Friedl P, Sebald W (1982) An Asp-Asn substitution in the proteolipid
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Isaev PI, Liberman EA, Samuilov VD, Skulachev VP, Tsofina LM (1970) Conversion of
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Jackson JB (2003) Proton translocation by transhydrogenase. FEBS Lett 545:1824
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193
Chapter 8
D
lH -Driven Mechanical Work: Bacterial
Motility
Minor ATPase activity of flagella has been discovered recently, but is was shown to be
necessary for the assembly of these organelles, and not for the movement per se.
2
Rotation of certain subunits of H+-ATP synthase (see preceding chapter) is used as a
mechanism of carrying out chemical (rather than mechanical) work.
195
196
D
lH -Driven Mechanical Work: Bacterial Motility
independently and the cell stops (or to be more precise, it starts performing short
chaotic movements in different directions (tumbling).
8.1 D
lH Powers the Flagellar Motor
In 1956, Peter Mitchell noted that it is theoretically possible to drive locomotion of
bacteria by an ionic gradient (Mitchell 1956). The bacterial flagellum was suggested to play the role of a giant ion channel. Later, this particular scheme was
ruled out, again theoretically, as ineffective. Nevertheless, a more general idea of
ion-driven locomotion was later proved experimentally.
In 1974, Julius Adler and colleagues (Larsen et al. 1974a) observed that an
E. coli mutant deficient in oxidative phosphorylation required respiration for
motility, although it could not be coupled to ATP synthesis because of the
mutation in the ATP synthase (the ATP level was maintained by glycolysis).
Moreover, a strong lowering of the ATP level did not stop the respiration-supported motility of the mutant, while the addition of an uncoupler did.
In 1975, experiments conducted by one of this books authors (V.P.S.) and his
coworker Kim Lewis confirmed the Adlers observation for quite another bacterium, Rhodospirillum rubrum. In this case too, bacterial motility did not correlate
with the ATP level (Glagolev and Skulachev 1978). But there was a good correlation between the rate of motility and the level of membrane potential produced
by the photosynthetic redox chain. On the basis of these observations, we suggested that the motility of Rh. rubrum is supported by D
lH , and not by ATP. Later,
three different laboratories (including the group of the books author) found that
bacteria paralyzed by exhaustion of endogenous sources for D
lH generation
become motile for several minutes when artificial DW or DpH is imposed
(Skulachev 1977; Matsura et al. 1977; Manson et al. 1977).
In Bacillus subtilis, motility appeared at about 30 mV D
lH and then increases
with increasing potential to 60 mV. Further increase in D
lH exerted no further
effect upon the locomotion rate. A D
lH threshold was also found in E. coli, but
here a 200 mV D
lH was necessary for the maximal speed to be obtained. This
maximal speed of locomotion varies for different species. The highest value
(140 lm/s) was observed for Bdellovibrio bacteriovorus (Stolp 1968). The
majority of other bacteria usually swim at speeds of 2040 lm/s. The flagellar
rotation rate is usually 1050 revolutions/s in the case of E. coli, reaching 300
revolutions/s when operating without load. In some other bacteria the flagellar
rotation rate can be as high as 1,700 revolutions per second.
An important feature of the flagellar motor is that it can rotate the flagellum
both clockwise and counterclockwise in spite of the fact that the D
lH direction
remains constant. This means that the motor is equipped with a gearshift. The
latter is controlled by the taxis system in such a way that repellents induce a
switch-over from counterclockwise to clockwise rotation, while attractants inhibit
this switch-over (Larsen et al. 1974b).
8.1 D
lH Powers the Flagellar Motor
197
In the case of certain bacteria, several different flagellin isoforms comprise the filament. The
physiological function of these isoforms is not yet clear.
4
Hereafter, the presented nomenclature of flagellum proteins is the one used for the bacterium
S. typhimurium.
198
D
lH -Driven Mechanical Work: Bacterial Motility
that a hollow tube is formed. The assembly of the filament involves the consecutive attachment of flagellin monomers from its distal end, which is located very
far away from the cell. Flagellin molecules are assumed to be delivered to the
distal end of the filament through the inner hollow tube of this structure, which
avoids substantial losses of the protein due to its diffusion to the outer medium.
The filament tube is covered on its far end by a special protein, FliD (another
name, HAP2), which prevents the depolymerization of flagellin monomers and
their diffusion into the outer medium. ATP hydrolysis by a special ATPase
composed of 6 FliI subunits and one FliH subunit (see below) is the driving force
of the flagellin-transport process. It is quite interesting that the amino acid
sequence of FliI is homologous to the a subunit of FoF1-ATP synthase, and six
subunits of this protein form a structure resembling the 3a : 3b-globule of factor F1
of the ATP-synthase complex (Imada et al. 2007). The FliH subunit is also
homologous to the b and d subunits of FoF1 ATP synthases (it is probably a
structural homolog of the peripheral stalk of the FoF1 complex), which implies the
use of a rotating mechanism for flagellin transport (see the preceding Sect. 7.1).
The filament is usually attached to the basal body by a hook, i.e., a special
curved structure of diameter somewhat larger than that of the filament. This hook
consists of a large number of copies of only one proteinFlgEattached to the
filament by two additional proteinsFlgL and FlgK (HAP1 and HAP3). The basal
body is a system of four (sometimes two) rings perpendicular to the filament and a
rod (comprising proteins FlgB, FlgC, FlgF, and FlgG) transpiercing the rings and
connected with the hook. The diameter of the rod varies from 8 to 12 nm, and that
of rings from 18 to 33 nm (Fig. 8.1ac).
The basal body of lateral flagella of gram-negative bacteria consists of four
rings. Rings L, P, M, and S were originally supposed to be part of the flagellum.
However, further research showed that rings M and S represent two different
domains of one protein, FliF, i.e., they form one ring, which was given the name
MS. Later, more careful extraction of the basal body revealed one more additional
ring that was found to be a part of this structure. This C ring is located on the
cytoplasmic side of the inner membrane (Fig. 8.1b). According to our current
understanding, there are four rings in the lateral basal body of flagella of gramnegative bacteriaL, P, MS, and C. The basal body of gram-positive bacteria
contains only two ringsMS and C.
The L ring is located inside the outer membrane and is composed of several
copies of lipoprotein FlgH. The P ring is located under the L ring and consists of
the FlgI protein. The MS ring is immersed in the cytoplasmic membrane and
consists of the FliF protein. The functions of all these rings are not yet known. The
C ring is located in the cytoplasm, and it consists of the proteins FlhA, FlhB, FliH,
FliJ, FliI, FliN, FliG, FliM, FliO, FliP, FliQ, and FliR. The proteins FliN, FliG, and
FliM are responsible at least for the switching of the rotation direction of the
flagellum (see below), while proteins FlhA, FlhB, FliH, FliJ, FliI, FliO, FliP, FliQ,
and FliR are required for the transmembrane transport of the flagellar proteins
during its assembly (see above).
199
Fig. 8.1 a A scheme of structure of the bacterial flagella basal body (for E. coli, C ring is not
shown); b electron micrograph of E. coli flagellum; arrows indicate (left to right) the basal body,
the rod, and the filament; the bar is 50 nm; the C ring is lost during the isolation procedure
(Skulachev 1988; Metlina 2001); c electron micrograph of basal body of the E. coli flagellum
(Berg 2000); d Mot complexes (arrow head) around basal body (arrow)
In the bacterial membrane one can see special protein structures surrounding
the basal body (Fig. 8.1d). They never accompany the basal body when it is
isolated from the cell. These structures are composed of a complex of integral
200
D
lH -Driven Mechanical Work: Bacterial Motility
membrane proteins MotA and MotB. The MotB protein forms one transmembrane
a-helix in its N-terminus, while its main part is located in the periplasm. A peptidoglycan-binding domain is located on the C-terminus of MotB. This protein is
supposed to be tightly bound to the bacterial cell wall. This fact suggests MotB to
be an element of the stator of the flagellar motor, for the stator of such a big
organelle capable of generating a torque of 4,000 picoNewton nanometers, has to
be firmly fixed in the only stiff bacterial structure, its cell wall.
There are four transmembrane a-helixes in the MotA protein, the rest of it (its
greater part) being located in the cytoplasm. MotA and MotB were shown to form
a stable complex. The MotAMotB complex is also known to interact with the
C-terminal domain of the FliG protein, which is part of the C ring. The number of
MotAMotB complexes in one flagellum has not yet been established. Structural
studies of various bacterial flagella provide the stoichiometry of 1016 MotAB
complexes per organelle, while functional studies of this motor predict the existence of eight torque generators.
Mutations in the transmembrane parts of MotA and MotB proteins lead to the
loss of D
lH -dependent rotation of the flagellum. MotA and MotB are considered
to be responsible for transmembrane proton transport; they are thought to be
stator components, while the basal body and the filament represent the rotor
part of the flagellar motor. The latter conclusion is supported by studies of the
consequences of genetic cross-linking of various protein components of the flagellum. Functionally active flagella were obtained with fused FliF-FliG and FliMFliN proteins, this fact being the basis for a conclusion about the role of MS and
C rings as rotor components of the organelle.
201
and C (rotor) and MotAB complexes (stator) form the functional part of the
bacterial motor.
In the stator there are supposed to be two proton wells crossing only part of
the hydrophobic membrane core. At the bottom of the inward proton well, there
has to be a proton-accepting group X, belonging to the rotor, i.e., to MS or C rings.
The appearance of a positively charged X on its protonated form results in its
coulombic attraction to a negatively charged proton acceptor group Y- (e.g. an
ionized carboxyl group of a dicarboxylic amino acid residue belonging to the
stator). This leads to the rotation of the rotor and a drawing together of X+H and
Y-. Proton transfer from X+H to Y- and further to the cytoplasm via the outward
proton channel completes the chain of events. Now a new H+ ion from the periplasm can protonate the next X group situated at the bottom of the inward H+ well.
The conservative aspartate residue of the only transmembrane a-helix of the
MotB protein (Asp32 in the case of E. coli) seems to be the best candidate for the
role of the Y group of the stator. Mutations of this amino acid lead to the complete
loss of D
lH -dependant rotation of the flagellum. However, it has not yet been
possible to find a good candidate for the role of the X group of the rotor. There are
no conservative residues of positively charged amino acids in the FliG, FliM, and
FliN proteins, and mutations of the charged amino acid residues in the C terminus
of the FliG protein (the domain interacting with the MotAB complex, see above)
do not result in the loss of the functional activity of the flagellum.
The above-suggested scheme has a number of other problems that remain to be
solved. For instance, this model assumes the existence of a long chain of groups
X at the periphery of the MS or C rings so that many protons are necessary to be
transferred across the membrane to cause a single turnover of the rotor. According
to calculations made by Berg, about 1,200 H+ ions should be transported across the
membrane to the cytoplasm per single rotor turnover (Berg et al. 1982). In the
framework of the scheme presented in Fig. 8.2, this means that the number of
X groups in the rotor should be about 1200/n, where n represents the number of
MotAB complexes in the flagellum, i.e., the number of X groups should be
Fig. 8.2 Possible arrangement of the bacterial proton motor (Glagolev and Skulachev 1978).
Explanations are provided in the text
202
D
lH -Driven Mechanical Work: Bacterial Motility
relatively high (*150), which is rather difficult to imagine if we take into account
the data obtained on the structure of the basal body.
In any case, the C ring seems to be better than the MS ring as the candidate for
the role of X group carrier. In this case there is no direct contact between rotor and
stator in the cytoplasmic membrane; it also helps to avoid the breakage of many
proteinprotein bonds in the hydrophobic membrane layer during rotor functioning. It would be easier to understand this mechanism if we postulate that rotor
stator interactions take place in the cytosol, while only the H+-ion transfer (causing
the change of the spatial location of the part of the H+-channel located in the
cytoplasmic part of the system) occurs in the membrane. In such a case, the rotor
rotation might take place in the membrane phospholipid layer without any contact
with those stator sites that are immersed in the membrane. In any other cases, there
appears the problem of the increased passive H+ leakage through the membrane in
the sites of the rotorstator contact.
As already mentioned, there is a gearshift in the flagellar motor allowing the
direction of rotation to be changed in spite of the constant direction of D
lH . It is
the C ring comprising the FliG, FliM, and FliN proteins that fulfills this function in
the organelle. The mechanism of this phenomenon should include the ability to
change the positions of the proton wells in the stator so that one of them would be
favorable for the clockwise and the other for counterclockwise rotation of the rotor
driven by proton transfer.
The movement of H+ ions along proton wells seems to be a physical process
since operation of the H+ motor does not depend on temperature. This may be
diffusion of H+ or H3O+ in water-containing pores. Another possibility is migration
of H+ along bound water molecules or along a chain of proteolytic groups without
any conformational changes in the protein molecule.
8.4 D
lH -Driven Movement of Non-Flagellar Motile
Prokaryotes and Intracellular Organelles of Eukaryotes
Besides the flagellar motor-driven movements, certain bacteria are capable of
other types of motility. The mechanisms of these alternative motility types have
not been thoroughly studied yet.
Experiments conducted in the group of one of this books authors (V.P.S.)
showed that the gliding motility of trichomes of the multicellular filamentous
cyanobacteria Phormidium uncinatum on the surface of a substrate is driven by
D
lH rather than ATP (Glagoleva et al. 1980; Skulachev 1980). This conclusion
was supported by inhibitor analysis of the gliding supported by photosynthetic,
respiratory, or glycolytic energy sources and by the fact that artificially imposed
DW or DpH were competent in supporting uncoupler-sensitive motility in the
presence of DCCD, which inhibited D
lH -powered ATP formation.
8.4 D
lH -Driven Movement of Non-Flagellar Motile Prokaryotes
203
204
D
lH -Driven Mechanical Work: Bacterial Motility
205
(up to 10 lm/s). Thus, the locomotive system used by these protozoa appears to be
very effective. The length of the devescovinid is about 0.1 mm, that of Mixotricha
paradoxa, about 0.5 mm. Some motile cyanobacteria can be 1 mm long.
No precedent has been described for D
lH to power the locomotion of a large
organism. Here, we always find an ATP-dependent mechanism of the actomyosin
type.
It is difficult to imagine a device in which a B20-lm-long bacterial flagellum is
employed to perform large-scale mechanical work necessary for locomotion of
multicellular animals. However, when studying the actomyosin mechanism, certainly very important for animals and humans, we should keep in mind that
diminutive forms of life have chosen quite a different mechanism for motility.
Accordingly, they had to invent a rotating ring; so, the wheel is not a human
invention. This ring is the heart of a more complex device, the basal body, which is
a molecular prototype of our electric motor (not a human invention either!) connected with a rigid filament (the screw of our motorboat).
This or a slightly modified engine is used not only to swim but also to glide by
cyanobacterial trichomes and even by some eukaryotic flagellates bearing symbiotic bacteria. In fact, these flagellates tried to build a steamer from many motor
boats. It is not surprising that such an attempt was a dead end of evolution.
References
Bardy SL, Ng SY, Jarrell KF (2003) Prokaryotic motility structures. Microbiology 149:295304
Berg HC (2000) Constraints on models for the flagellar rotary motor. Philos Trans R Soc Lond B
Biol Sci 355:491501
Berg HC, Manson MD, Conley MP (1982) Dynamics and energetics of flagellar rotation in
bacteria. Symp Soc Exp Biol 35:131
Bredt W (1973) Motility of mycoplasmas. Ann NY Acad Sci 225:246250
Cleveland LR, Grimstone AV (1964) The fine structure of the flagellate Mixotricha paradoxa and
its associated micro-organisms. Proc R Soc Lond B 159:668686
Eisenbach M, Adler J (1981) Bacterial cell envelopes with functional flagella. J Biol Chem
256:88078814
Glagolev AN, Skulachev VP (1978) The proton pump is a molecular engine of motile bacteria.
Nature 272:280282
Glagoleva TN, Glagolev AN, Gusev MV, Nikitina KA (1980) Proton motive force supports
gliding in cyanobacteria. FEBS Lett 117:4953
Halfen LN, Castenholz RW (1970) Gliding in a blue-green algae: a possible mechanism. Nature
225:11631165
Halfen LN, Castenholz RW (1971) Gliding motility of blue-green algae: Oscillatoria princeps.
J Phycol 7:133145
Imada K, Minamino T, Tahara A, Namba K (2007) Structural similarity between the flagellar
type III ATPase FliI and F1-ATPase subunits. Proc Natl Acad Sci U S A 104:485490
Khan S, Berg HC (1983) Isotope and thermal effects in chemiosmotic coupling to the flagellar
motor of Streptococcus. Cell 32:913919
Larsen SH, Adler J, Gargus JJ, Hogg RW (1974a) Chemomechanical coupling without ATP: the
source of energy for motility and chemotaxis in bacteria. Proc Natl Acad Sci U S A 71:1239
1243
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D
lH -Driven Mechanical Work: Bacterial Motility
Larsen SH, Reader RW, Kort EN, Tso WW, Adler J (1974b) Change in direction of flagellar
rotation is the basis of the chemotactic response in Escherichia coli. Nature 249:7477
Manson MD, Tedesco P, Berg HC, Harold FM, Van der Drift C (1977) A protonmotive force
drives bacterial flagella. Proc Natl Acad Sci U S A 74:30603064
Manson MD, Tedesco PM, Berg HC (1980) Energetics of flagellar rotation in bacteria. J Mol Biol
138:541561
Matsura S, Shioi J, Imae Y (1977) Motility in Bacillus subtilis driven by an artificial
protonmotive force. FEBS Lett 82:187190
Metlina AL (2001) Procariotic flagella as system of biological motility. Usp Biol Chem 41:229
282 (in Russian)
Mitchell P (1956) Hypothetical thermokinetic and electrokinetic mechanisms of locomotion in
microorganisms. Proc R Phys Soc Edinb 25:3234
Skulachev VP (1977) Transmembrane electrochemical H+-potential as a convertible energy
source for the living cell. FEBS Lett 74:19
Skulachev VP (1980) Membrane electricity as a convertible energy currency for the cell. Can J
Biochem 58:161175
Skulachev VP (1988) Membrane bioenergetics. Springer, Berlin
Stolp H (1968) Bdellovibrio bacteriovorusa predatory bacterial parasite. Naturwissenschaften
55:5763 (in German)
Tamm SL (1982) Flagellated ectosymbiotic bacteria propel a eucaryotic cell. J Cell Biol 94:697
709
Waterbury JB, Willey JM, Franks DG, Valois FW, Watson SW (1985) A cyanobacterium capable
of swimming motility. Science 230:7476
Chapter 9
D
lH -Driven Osmotic Work
cell, D
lNa - or ATP-driven transport is much more typical, but in certain, in fact
rather special cases, they can be powered by D
lH . In bacteria, one can find all the
instead of D
lH . For instance, in E. coli, among 16 systems for uphill transport of
amino acids there are 8 utilizing ATP, 5 utilizing D
lH , 1 utilizing D
lNa , and 2
supported by D
lH and D
lNa (Table 9.1). In the same E. coli, glucose and lactose
transports are mediated by PEP- and D
lH -dependent systems, respectively.
For solute S, accumulated by a D
lH -driven system in a negatively charged
compartment (e.g. in the mitochondrial matrix or the bacterial cytoplasm) the
accumulation ratio ([S]in/[S]out) will obey the equation:
RT ln
Sin
H
n Z) F DW nRT ln in ;
Sout
H out
9:1
207
9 D
lH -Driven Osmotic Work
208
Table 9.1 Driving forces for
accumulation of amino acids
in E. coli cells
System number
Amino acid
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Ala, Gly
Thr, Ser
Leu, Ile, Val
Leu, Ile, Val
Leu, Ile, Val
Leu, Ile, Val
Phe, Tyr, Trp
Pro
Lys, Arg, Orn
Arg
Gln
Gln
Gln
Gln
Cys
Cys
D
lH
D
lH
ATP
ATP
D
lH
D
lH
D
lH
D
lH , D
lNa
ATP
ATP
ATP
ATP
lNa
D
lH , D
D
lH , D
lNa
ATP
ATP
where n is the number of H+ ions symported with one S molecule, and Z is the
number of positive charges on S.
C Z in
Z F DW:
C Z out
9:2
On the basis of the Nernst equation, it is easy to calculate that a tenfold gradient
of a monovalent cation C+ requires a DW of about 60 mV to be formed (at 25 C).
Such simple electrophoretic behavior characterizes the system of valinomycinmediated transport of K+ ions. A similar mechanism is responsible for Ca2+
accumulation by mitochondria and bacteria (Fig. 9.1., system 1). The only difference is that in this case coefficient Z is equal to 2, so that 30 mV DW is sufficient
to maintain the tenfold gradient.
Both valinomycin and mitochondrial Ca2+-uniporter have a rather simple
problem to solve. They should recognize the transported ion and facilitate its
diffusion across the hydrophobic membrane core. No special device to transduce
DW energy into an ion concentration gradient is necessary, since the overall
process consists of the movement of the ion in the electric field (electrophoresis).
209
9 D
lH -Driven Osmotic Work
210
9:3
The simplest case is when a weak acid or a weak base is accumulated in the
more alkaline or more acidic compartment, respectively. To do this, only two
conditions should be fulfilled: the membrane should be permeable for the
uncharged form of the acid (base), and it must be impermeable for the charged
form of this acid (base). Accordingly, the following simple events described by
Eqs. (9.4) and (9.5) occur:
AHout ! AHin;
9:4
AHin OH
in ! Ain H2 Oin:
OH
in
9:5
+
Consumed
is regenerated by the D
lH generators removing H from, e.g.,
the mitochondrial matrix.
Exactly in this way, acetate is taken up by energized mitochondria. Since acetic
acid in its protonated form, CH3COOH, is a small uncharged molecule, it enters
mitochondria without any carriers, moving down DpH, neutralizes intramitochondrial OH- ions, and accumulates inside in the form of the acetate anion
(CH3COO-). The latter is charged and thus cannot cross the mitochondrial
membrane and escape from the matrix.
A similar mechanism, but operating in the opposite direction, can give rise to
NH3 efflux:
211
9:6
NH3 out H
out ! NH4
out
9:7
For weak acids or bases that are lipid-insoluble, corresponding carriers are
required that are specific to the uncharged form of the solute and incapable of
transporting its anion or cation. This mechanism is exemplified by a mitochondrial
succinate carrier which binds succinate2- (-OOC-CH2-CH2-COO-) on the outer
surface of the inner membrane and symports it with 2 H+out into the matrix space.
This is equivalent to uniport of undissociated succinic acid (HOOC-CH2-CH2COOH), the concentration of which in neutral solutions is very low (see above,
Fig. 9.1, system 2).
Thus, the existence of D
lH in its secondary form (DpH) allows weak acids and
bases to be involved in uphill transport in the direction determined by the pH
gradient. Yet just as in the case of the DW-driven transport of cations and anions, it
is hardly possible to obtain a reasonable composition of, e.g., the bacterial cytoplasm by accumulation of anions of weak acids and cations of strong bases, and by
export of cations of weak bases and anions of strong acids. It seems obvious that in
some cases the direction of the solute fluxes must be opposite.
For cations of strong bases, the problem can be solved by using cation/H+antiporters. For example, mitochondria (Williams and Fry 1979) and some bacteria
(Zimniak and Barnes 1980), in addition to the electrophoretic system for Ca2+
accumulation, also have an electroneutral Ca2+/2H+-antiporter exporting Ca2+
from the mitochondrial matrix or the bacterial cytoplasm down DpH (Fig. 9.1,
system 3).
Thus, mitochondria and bacteria can not only accumulate Ca2+ but also actively
export it. It is clear that these two systems must be alternatively actuated since
their cooperation would result in Ca2+ circulation dissipating energy of D
lH .
Similarly, anions of strong acids can be accumulated inside the matrix in a
DpH-dependent manner if they are symported with H+. This can be exemplified by
phosphate transport systems of mitochondria and bacteria. Electroneutral H3PO4 is
practically absent from aqueous solution at physiological pH. Phosphate anions
must be exported from the mitochondrial matrix and bacterial cytoplasm down
DW. Nevertheless, phosphate is accumulated, not exported. This results from the
operation of the carrier that symports H2PO4- with H+.
9.4 Total D
lH As Driving Force
Sometimes both DW and DpH are driving forces for uphill transport of a solute.
This is the case, for instance, when the transported compound does not contain
either ionized atoms or mobile protons. Such a compound can be symported with
H+ (or antiported against H+) down total D
lH . Solute, H+-symport is responsible
212
9 D
lH -Driven Osmotic Work
for the accumulation of lactose in bacteria (Fig. 9.1, system 4; for details, see Sect.
9.7.1). Vacuoles of sugar beet taproot were shown to accumulate sucrose in a
D
lH -driven manner, the process being mediated by sucrose/H+-antiporter (Briskin et al. 1985). The reason for using an antiporter instead of a symporter is that
the directions of D
lH in the vacuole and in a bacterium are opposite.
For lactose, H+-symport, as well as for sucrose/H+-antiport, DW and DpH
should be equally effective as the driving forces. In such cases, an increase in the
H+/solute stoichiometry simply enhances the gradient of the substance resulting
from the transport process. In other cases, an increase in stoichiometry is required
to obtain the necessary direction of transport. Accumulation of the positively
charged catechol amines (RNH3+) in chromaffin granules of adrenal medulla is an
example. Here, H+-ATPase pumps H+ into the granules, so that their interior is
charged positively and acidified (see Sect. 9.7.2). When accumulated in granules,
RNH3+ is antiported against 2 H+ (see Fig. 9.1, system 5). So, import of one
RNH3+ into the granule interior is coupled to the transport of two protons and only
one net positive charge in the opposite directions. These data suggest that at equal
DW and DpH, the contribution of DpH will be twofold larger than that of DW
(see above, Eq. (9.1)). On the other hand, in systems like TrkA (see Sect. 9.2)
when a cation (K+) is symported with H+, the contribution of DW is twofold larger
than DpH since the transport of one K+ is accompanied by the movement of two
net positive charges (K+ and H+) and only one proton.
As long as the transport processes are reversible, the metabolically generated
gradient of a solute exported from the cell by a D
lH -linked system can, in
principle, be an energy source for D
lH formation. For example, in anaerobic
Enterococcus hirae, glycolytic lactate production results in the formation of a
lactate gradient ([lactate]in[[lactate]out). The efflux of lactate was found to be
catalyzed by lactate, nH+-symporter where n is equal to 2 at low [lactate]out and
[H+]out, and is equal to 1 when the lactate and H+ levels in the medium increase
(Simpson et al. 1983). This means that cooperation of glycolysis and the symporter
alkalinizes the cytoplasm and charges it negatively when outer concentrations of
lactate and protons are low:
glucose ! 2 lactate
in 2 Hin ;
9:8
2 lactate
in 4 Hin ! 2 lactateout 4 Hout :
9:9
9.5
D
lH -Driven Transport Cascades
213
9.5 D
lH -Driven Transport Cascades
Several systems have been described where D
lH powers the formation of a gradient of a solute (S1) which is then used for uphill transport of another solute (S2).
In bacteria, the role of the S1 intermediate can be performed by Na+. In this
+
+
case, D
lH drives electroneutral Na+in =H
out or electrogenic Nain/nH out-antiport so
that the formed DpNa can be used to drive accumulation of various metabolites in
the cytoplasm by means of Na+out, solute-symporters. This is the main type of
osmotic work in alkalophilic, halophilic, and many marine microorganisms.
In mitochondria, a long D
lH -dependent cascade was described for the transport of phosphate and Krebs cycle intermediates. The initial event is DW formation
by the respiratory chain. Then DW is discharged by a flux of ionized penetrants
2+
+
3
(electrophoretic ATP4
in =ATPout exchange, Ca - or K -uniport, etc.) and DpH is
formed. The DpH is utilized to form DpPi via H2PO4 , H+-symport directed from
the outside to the inside. The DpPi powers the accumulation of malate by means of
an HPO42-/malate2--antiporter. (The same carrier can also transport some other
dicarboxylates.) The last step of the cascade is import of citrate via malate2-/
citrate3-,H+-antiporter (Fig. 9.2). Such a complicated system allows a very
sophisticated regulation of the mitochondrial metabolic pattern to be organized.
214
9 D
lH -Driven Osmotic Work
215
Fig. 9.3 Carnitine and acylcarnitine in deprotonated (zwitterion) and protonated (cationic)
forms. R, a fatty acyl
membrane (Fritz 1963). This idea was supported by the findings that (i) fatty
acylcarnitine transferase, catalyzing acyl transfer between carnitine and CoA, is
localized on both sides of the hydrophobic barrier of the inner mitochondrial
membrane, and (ii) this membrane is permeable for fatty acylcarnitine, but not for
fatty acyl-CoA (Bremer 1983). Thus, the function of carnitine (Cn) as the fatty
acyl carrier was summarized in the following chain of events (Eqs. (9.109.12)):
acylCoAout Cnout ! acyl Cnout CoAout ;
9:10
9:11
9:12
216
9 D
lH -Driven Osmotic Work
acylCnout H
out ! acylCnHout ;
9:13
acylCnH
out ! acylCnHin ;
9:14
acylCnH
in ! acylCnin Hin :
9:15
In agreement with this scheme, Dmitry Levitsky found that a planar phospholipid membrane is permeable for the palmitoylcarnitine cation (Levitsky and
Skulachev 1972). As subsequent experiments showed, a discharge of the mitochondrial membrane potential by uncouplers strongly inhibited the oxidation of
palmitoylcarnitine added after the uncoupler. On the other hand, the uncoupler
stimulated oxidation of palmitoylcarnitine when the latter was added 3 min before
the uncoupler. Apparently palmitoyl carnitine accumulated inside energized
mitochondria during these 3 min in an amount sufficient to support active respiration during the entire time of the polarographic experiment.
Thus, there came the answer to the question why it is carnitine that plays the
role of a fatty acyl carrier. All three functional groups of carnitine are necessary
for the function of fatty acyl, H+-symporter: hydroxyl, to combine with the fatty
acyl residue; carboxyl, to reversibly bind the H+ ion; and quaternary nitrogen, to
charge protonated acylcarnitine positively (see Fig. 9.3).
This concept failed to answer one final question: what is the fate of the intramitochondrial carnitine released in the reaction with CoAin? This problem was
solved in 1976 by Shri Pande and Rehana Parvin and independently by Rona
Ramsay and Philip Tubbs, who found stoichiometric carnitine/fatty acylcarnitine
antiport across the inner mitochondrial membrane (Pande and Parvin 1976;
Ramsay and Tubbs 1976).
A general scheme of events resulting in the transport of extramitochondrial
fatty acids into the matrix is given in Fig. 9.4. The process starts in the outer
mitochondrial membrane where acyl-CoA synthase is operating. This enzyme
esterifies CoA with fatty acids at the expense of the energy of ATP (reaction 1). In
the intermembrane space or on the outer surface of the inner membrane, acyl-CoA
is attacked by outer fatty acylcarnitine transferase so that acyl is transferred from
CoA to carnitine (reaction 2). The resulting acylcarnitine is protonated by H+out.
The pool of H+out is regenerated by respiratory D
lH generators that pump protons
from the matrix to the intermembrane space (reactions 3 and 4). The protonated
acylcarnitine cation moves across the inner membrane down DW and DpH
(reaction 5) and is deprotonated in the matrix (reaction 6). Acylcarnitine transfers
its acyl group to intramitochondrial CoA, this being catalyzed by the inner fatty
acylcarnitine transferase (reaction 7). The formed inner carnitine is exchanged for
outer acylcarnitine via the carnitine/acylcarnitine-antiporter (reaction 8).
An interesting feature of this system is that the level of the matrix carnitine
regulates the distribution of the flow of acylcarnitine between the D
lH -driven (i.e.
energy-expending) pathway 5 and the energy-independent antiporter-mediated
217
Fig. 9.4 Carnitine-mediated transport of fatty acyl groups into the mitochondrial matrix: 1 acylCoA synthase; 2 outer carnitine acyltransferase; 3 D
lH generators of the respiratory chain; 4
protonation of the acylcarnitine carboxyl group; 5 D
lH -driven influx of protonated acylcarnitine;
6 deprotonation of the acylcarnitine carboxyl group; 7 inner carnitine acyltransferase; 8 carnitine/
acylcarnitine-antiporter
218
9 D
lH -Driven Osmotic Work
exported from mitochondria down DpH even without any carriers because mitochondrial membranes are permeable to CH3COOH and NH3. The carrier only
accelerates these fluxes. On the other hand, the involvement of a carrier is quite
necessary when the transported solute or chemical groups per se cannot move
across the membrane in the proper direction. The living cell always tries to avoid
spontaneous events, preferring to deal with protein-mediated processes to achieve
a high specificity and a controlled reaction rate that can be regulated within wide
limits, from very high down to zero values. This principle can be applied, in
particular, to D
lH -linked osmotic work. The great majority of transport mechanisms are carrier mediated.
In rare cases, these carriers are very simple, such as carnitine. Such peptide
antibiotics as channel-forming gramicidin that transports Na+, K+, and H+, as well
as the K+-uniporter valinomycin, K+/H+-antiporter nigericin, or Na+/H+-antiporter
monensin also look like rather simple molecules in comparison with proteins.
However, one should remember that both carnitine and the above-mentioned
antibiotics perform, in fact, primitive functions.
In the case of carnitine, it is the initiation of a process that then becomes
mediated by a protein, namely, the carnitine/acylcarnitine-antiporter. The role of
antibiotics is apparently restricted to damaging cell membranes of species other
than the producer. When the function of a carrier is more complicated, it is always
of protein nature.
The carrier proteins are integral membrane polypeptides that may be assisted
by peripheral or water-soluble proteins. In the above-mentioned well-studied
example of E. coli amino acid transport (see Table 9.1), it was shown that porters
1, 2, 5, 7, 8, and 14 do not require any assistants. In the other systems listed in
Table 9.1, there are specific transport-assisting proteins in the periplasm. In systems 3, 4, and 6, these proteins act as buffers of the transported amino acids. They
reversibly bind these amino acids, in this way sequestering them in the periplasm.
As a result, the import of amino acids into the cell does not cause an immediate
decrease in the amino acid concentration in the periplasm. This should stabilize the
import rate.
Another situation is found in the case of systems 9 and 10. Here, the assisting
proteins are necessary for transport per se. They bind corresponding amino acids in
the periplasm and then form a complex with an integral protein of the cytoplasmic
membrane, which plays, in fact, the role of apo-carrier. For systems 9 and 10, it
is the assisting protein that determines the specificity and affinity of the carrier to
the transported solute. Below we will consider some of the D
lH -driven transport
systems that have been studied in detail.
219
220
9 D
lH -Driven Osmotic Work
Fig. 9.5 Structure of lactose, H+-symporter from E. coli in a complex with substrate analog
(black) (Abramson et al. 2003). a side view (parallel to the membrane surface); b top view (from
the cytosol side)
Fig. 9.6 Structural
differences between the open
inward a and open outward
b conformations of lactose,
H+-symporter. The substrate
analog is colored black
(Abramson et al. 2003)
221
222
9 D
lH -Driven Osmotic Work
same is true for substrate-level phosphorylation coupled to oxidative decarboxylation of a-ketoglutarate, which forms GTP. This means that by inhibiting the
matrix nucleoside diphosphate kinase, a mitochondrion can prevent its GTP pool
from being exhausted by extramitochondrial energy consumers. This should also
facilitate protein synthesis reactions in mitochondria, for they are supported by the
energy of GTP.
The ATP/ADP-antiporter is a hydrophobic membrane protein (molecular mass,
32 kDa) comprises 297 amino acid residues (in case of the bovine enzyme).
According to the analysis of its amino acid primary sequence, it consists of three
repeating sequences, each of them comprises two hydrophobic a-helices connected
by a long hydrophilic loop (i.e. there are six transmembrane a-helices in the
complete protein). Homologous repeats in the structure of this carrier indicate its
most probable origin: it must have appeared in evolution as a result of triplication
of some precursor gene. The antiporter operates as a homodimer in vivo.
The three-dimensional structure of the ATP/ADP-antiporter has been recently
determined by X-ray analysis (Pebay-Peyroula et al. 2003). Six transmembrane
segments of this protein form a rather compact structure in the lipid bilayer
(Fig. 9.9). All of these a-helices are substantially bent in relation to the axis
perpendicular to the membrane surface. Each second a-helix has an acute distortion approximately in the middle of the bilayer due to the presence of proline
residues. Similar to lactose permease, there is a large hydrophilic cone in the
protein (Fig. 9.10). It is surfaced on the inside with positively charged amino acid
residues (Arg79, Arg104, Arg137, Arg234, Arg235, Arg279; Lys22, Lys32,
Lys91, Lys95, and Lys106), which probably provide recognition, release from
magnesium, and binding of ATP4- and ADP3-. The binding site of these
223
nucleotides is located at the very bottom of this positively charged cavity (i.e.
around the middle of the lipid bilayer). At this site, the protein has an unusual
sequence (Arg234Arg235Arg236Met237Met238Met239) that is characteristic for the
primary structure of ATP/ADP-antiporters and is absent from other carriers.
The mechanism of nucleotide exchange by ATP/ADP-antiporter is probably
similar to the mechanism of the above-described lactose, H+-symporter. This
mechanism could embrace substantial conformational changes of the protein
during of its functioning; these changes might result in the opening of the
hydrophilic cavity either in the direction of the matrix (this variant of the protein
structure should have an increased affinity to ATP), or in the direction of the
intermembrane space (accompanied by simultaneous decrease in affinity to ATP
and increase in affinity to ADP). It is important to note that ATP/ADP-antiporter
probably catalyzes nucleotide exchange only when in the form of a homodimer.
224
9 D
lH -Driven Osmotic Work
Thus, the possible mechanism of functioning of this homodimer includes cooperative change in monomer conformations with hydrophilic cavities of these
monomers opening to the opposite membrane sides.
The ATP/ADP-antiport is found not only on the inner mitochondrial membrane.
An indication that ATP/ADP-antiport occurs in chloroplasts was first obtained by
Woldegiorgis and colleagues (1985). Nucleotide exchange in chloroplasts is sensitive to bongkrekic acid, but resistant to carboxyatractylate. The plastid antiporter
was shown to function in the direction opposite to the one of mitochondria, i.e., it
exchanges cytoplasmic ATP for the ADP in the organelle, thus providing for
chloroplast functioning in the dark.
A special system of adenine nucleotide transfer was discovered in the cytoplasmic membrane of Rickettsia prowazekii, an intracellular parasitic bacterium of
eukaryotic cells and epidemic typhus pathogen. The bacterium has a respiratory
chain that maintains D
lH across its membrane. However, the ATP produced by
the host cell was also found to be an energy source for Rickettsia due to the
presence of a special ATP/ADP-antiporter in the bacterium. This protein provides
ATP influx into the microorganism and ADP efflux from it, i.e., the direction of
nucleotide transport in this case is opposite to the one observed in mitochondria
(Andersson 1998). These data explain the fact that the ATP/ADP-antiporter of this
microorganism is much more similar to the antiporter of chloroplast type than to
its mitochondrial analog (even though R. prowazekii and mitochondria are close
evolutionary relatives) and shed light on the evolutional mechanism of the
origin of mitochondria.
An ATP-import mechanism has also been described for Bdellovibrio bacteriovorus (Ruby and McCabe 1986). This small bacterium has the highest rate of
motility among prokaryotes. Swimming very fast, it kills other microorganisms by
breaking their cell wall. When it finds itself inside a cell, B. bacteriovorus imports
the ATP of the sacrificed cell.
9.8 Role of D
lH in Transport of Macromolecules
One of the most intriguing problems of membranology and bioenergetics is how
biological macromolecules, i.e., proteins and nucleic acids, can be transported
across the hydrophobic barriers of coupling membranes impermeable even to such
small particles as H+ ions.
It is firmly established that such processes of macromolecules transport occur
without membrane breakdown since it was found that in many cases that to proceed with a normal rate they need D
lH or one of its constituents. This rate, at least
in some cases, is very high.
Two models of protein transport across the coupling membranes have been
most thoroughly studied: the import of mitochondrial (chloroplast) proteins synthesized in cytosol, and the export of bacterial periplasmic or outer membrane
proteins.
9.8 Role of D
lH in Transport of Macromolecules
225
226
9 D
lH -Driven Osmotic Work
inner membrane through a channel formed by the Tim23 subunit. The latter
process occurs due to electrophoretic transfer of positively charged amino acid
residues of the leader, i.e. it is DW-driven. The absence of DW on the inner
mitochondrial membrane completely prevents the transport of mitochondrial
matrix proteins.
When in the matrix, the leader sequence binds with the chaperone mtHsp70,
which (together with additional proteins) fulfills two functions. The chaperone
drags down the rest of the precursor protein into the matrix at the expense of
energy of ATP hydrolysis, and then it secures the correct assembly of the mature
mitochondrial protein.
Thus, protein transport to the mitochondrial matrix requires energy in the form
of both DW and ATP. However, when proteins are transported from the cytosol to
inner mitochondrial membrane, it is only energy of DW that is utilized.
There are a small number of very hydrophobic polypeptides coded by mitochondrial genome and synthesized in the matrix: seven subunits of NADH:CoQoxidoreductase, one subunit of CoQH2:cytochrome c-oxidoreductase, three subunits of cytochrome oxidase, and two subunits of H+-ATP-synthase.1 All of these
are synthesized as mature forms (without any precursors involved) and are
incorporated into the membrane by a special complex different from the TIM
complex. The mechanism of this process remains unclear.
An important conclusion from the above-discussed data is that a mitochondrion
cannot be formed by self-assembly from its constituents. It can originate only from
another mitochondrion, just as a daughter cell is formed from a mother cell.
A necessary condition for mitochondrial biogenesis is a DW across its inner
membrane. Since no mitochondrial DW generator can be formed solely from
mitochondrially synthesized polypeptides, a mitochondrion that has lost its last
DW-generating enzyme cannot make up for this deficiency even if the surrounding
cytoplasm contains an excess of mitochondrial protein precursors. This may be the
reason why yeasts growing under anaerobic conditions retain closed vesicles,
promitochondria, containing no respiratory chain but competent in DW formation
mediated by reverse H+-ATP-synthase, small amounts of which are present there.
9.8 Role of D
lH in Transport of Macromolecules
227
228
9 D
lH -Driven Osmotic Work
(pilot) proteins so that large molecular masses cross the hydrophobic barrier. In
some cases, double-stranded DNA was found to be transported (Grinius 1986).
The role of D
lH and ATP in DNA transport was studied by Leonas Grinius
and colleagues. They found, in particular, that the exhaustion of the intracellular
ATP pool by arsenate does not inhibit either T4-phage infections or genetic
transformation (Kalasauskaite et al. 1980). On the other hand, conjugation is
inhibited by arsenate. As to D
lH , it was necessary for all three types of bacterial
DNA transport processes. This was shown for Bacillus subtilis transformation,
E. coli T4-phage infection, and conjugation.
Later these observations were confirmed in other laboratories. In particular,
Wout Bremer et al. found that transformation-inducing DNA transport into
B. subtilis or Haemophilus influenzae cells is D
lH -dependent, both DW and DpH
being effective (Bremer et al. 1984). Bernard Labedan et al. confirmed that D
lH is
essential for T4-phage infection but pointed out that only DW, and not DpH, is
effective (Labedan and Goldberg 1979; Labedan et al. 1980). Labedans group,
studying a T5 phage-induced infection, failed to observe the requirement for D
lH .
The transport of the k-phage DNA into E. coli was also found to be energyindependent. Apparently, bacteria possess several mechanisms for phage DNA
transport across the membrane. In cases when D
lH is necessary, it may be a
driving force and/or a regulator essential for the correct orientation of the transported DNA or DNAprotein complex.
References
Abramson J, Smirnova I, Kasho V, Verner G, Iwata S, Kaback HR (2003) The lactose permease
of Escherichia coli: overall structure, the sugar-binding site and the alternating access model
for transport. FEBS Lett 555:96101
Adrian GS, McCammon MT, Montgomery DL, Douglas MG (1986) Sequences required for
delivery and localization of the ADP/ATP translocator to the mitochondrial inner membrane.
Mol Cell Biol 6:626634
Andersson SG (1998) Bioenergetics of the obligate intracellular parasite Rickettsia prowazekii.
Biochim Biophys Acta 1365:105111
Bogdanov M, Xie J, Dowhan W (2009) Lipid-protein interactions drive membrane protein
topogenesis in accordance with the positive inside rule. J Biol Chem 284:96379641
Bremer J (1983) Carnitinemetabolism and functions. Physiol Rev 63:14201480
Bremer W, Kooistra J, Hellingwerf KJ, Konings WN (1984) Role of the electrochemical proton
gradient in genetic transformation of Haemophilus influenzae. J Bacteriol 157:868873
Briskin DP, Thornley WR, Wyse RE (1985) Membrane transport in isolated vesicles from
sugarbeet taproot: II. evidence for a sucrose/H+-antiport. Plant Physiol 78:871875
Carter HE, Bhattacharyya PK, Weidman KR, Fraenkel G (1952) Chemical studies on vitamin BT
isolation and characterization as carnitine. Arch Biochem Biophys 38:405416
Cohen GN, Rickenberg HV (1955) Direct study of the fixation of an inductor of betagalactosidase by Escherichia coli cellules. C R Hebd Seances Acad Sci 240:466468
Ehrenstein G, Lecar H (1977) Electrically gated ionic channels in lipid bilayers. Quart Rev
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Epstein W, Laimins L (1980) Potassium transport in Escherichia coli: diverse systems with
common control by osmotic forces. Trends Biochem Sci 5:2123
Friedman S, Fraenkel G (1955) Reversible enzymatic acetylation of carnitine. Arch Biochem
Biophys 59:491501
Fritz I (1955) The effect of muscle extracts on the oxidation of palmitic acid by liver slices and
homogenates. Acta Physiol Scand 34:367385
Fritz IB (1963) Carnitine and its role in fatty acid metabolism. Adv Lipid Res 1:285334
Grinius LL (1986) Bacterial transport of macromolecules. Nauka, Moscow
Gulewitsch W, Krimberg R (1905) Zur kenntnis der extraktivstoffe der muskeln. 2 Mitteilung.
ber das Carnitin. Hoppe-Seylers Z Physiol Chem 45:326330
Kaback HR (1983) The lac carrier protein in Escherichia coli. J Membr Biol 76:95112
Kaback HR, Wu J (1997) From membrane to molecule to the third amino acid from the left with a
membrane transport protein. Q Rev Biophys 30:333364
Kalasauskaite E, Grinius L, Kadisaite D, Jasaitis A (1980) Electrochemical H+ gradient but not
phosphate potential is required for Escherichia coli infection by phage T4. FEBS Lett
117:232236
Klingenberg M, Rottenberg H (1977) Relation between the gradient of the ATP/ADP ratio and
the membrane potential across the mitochondrial membrane. Eur J Biochem 73:125130
Labedan B, Goldberg EB (1979) Requirement for membrane potential in injection of phage T4
DNA. Proc Natl Acad Sci U S A 76:46694673
Labedan B, Heller KB, Jasaitis AA, Wilson TH, Goldberg EB (1980) A membrane potential
threshold for phage T4 DNA injection. Biochem Biophys Res Commun 93:625630
Levitsky DO, Skulachev VP (1972) Carnitine: the carrier transporting fatty acyls into
mitochondria by means of an electrochemical gradient of H+. Biochim Biophys Acta
275:3350
Maccecchini ML, Rudin Y, Blobel G, Schatz G (1979) Import of proteins into mitochondria:
precursor forms of the extramitochondrially made F1-ATPase subunits in yeast. Proc Natl
Acad Sci U S A 76:343347
Mitchell P (1963) Molecule, group and electron translocation through natural membranes.
Biochem Soc Symp 22:142169
Mller M (1992) Proteolysis in protein import and export: signal peptide processing in eu- and
prokaryotes. Experientia 48:118129
Newman MJ, Wilson TH (1980) Solubilization and reconstitution of the lactose transport system
from Escherichia coli. J Biol Chem 255:1058310586
Pande SV, Parvin R (1976) Characterization of carnitine acylcarnitine translocase system of heart
mitochondria. J Biol Chem 251:66836691
Pebay-Peyroula E, Dahout-Gonzalez C, Kahn R, Trzguet V, Lauquin GJ, Brandolin G (2003)
Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside. Nature
426:3944
Pivetti CD, Yen MR, Miller S, Busch W, Tseng YH, Booth IR, Saier MH Jr (2003) Two families
of mechanosensitive channel proteins. Microbiol Mol Biol Rev 67:6685
Ramsay RR, Tubbs PK (1976) The effects of temperature and some inhibitors on the carnitine
exchange system of heart mitochondria. Eur J Biochem 69:299303
Ruby EG, McCabe JB (1986) An ATP transport system in the intracellular bacterium,
Bdellovibrio bacteriovorus 109 J. J Bacteriol 167:10661070
Schatz G (1993) The protein import machinery of mitochondria. Protein Sci 2:141146
Schnheit P, Beimborn DB, Perski H-J (1984) Potassium accumulation in growing Methanobacterium thermoautotrophicum and its relation to the electrochemical proton gradient. Arch
Microbiol 140:247251
Severin SE, Skulachev VP, Yaguzhinskiy LS (1970) Possible role of carnitine in the transport of
fatty acids through the mitochondrial membrane. Biokhimiia 35:12501253
Simpson SJ, Bendall MR, Egan AF, Vink R, Rogers PJ (1983) High-field phosphorus NMR
studies of the stoichiometry of the lactate/proton carrier in Streptococcus faecalis. Eur J
Biochem 136:6369
230
9 D
lH -Driven Osmotic Work
Chapter 10
D
lH as Energy Source for Heat
Production
231
232
10
D
lH as Energy Source for Heat Production
The latter concept seems to be more reasonable since the aim of the heat regulation
mechanism is to make physiological functions independent of the temperature of
the medium.
10.2
233
234
10
D
lH as Energy Source for Heat Production
Fig. 10.1 Cold-induced thermogenesis in brown fat mitochondria. Symbols + and - stand
for stimulation and inhibition, respectively. For explanation, see the text
10. The H+ ions return to the matrix moving downhill. This is mediated by the
fatty acidthermogenin system. This final event results in heat production.
Two alternative hypothetical mechanisms of the uncoupling effect of UCP have
been suggested.
According to one, binding of fatty acids to this protein opens a channel in it that
allows transmembrane H+ transfer (Klingenberg 2001).
An alternative mechanism was suggested by one of this books authors (V.P.S.).
According to this hypothesis, thermogenin is a carrier of the anion form of free
fatty acids (Skulachev 1991, 1998, 1999). Facilitation of transmembrane transfer
of these anions (RCOO-) can play a significant role in the uncoupling effect of
fatty acids, for their anion forms are stopped at the membrane/water interface
while facing water with their carboxyl residue and facing lipid with the fatty
tail. That is why membranes, while being permeable for neutral (protonated)
10.2
235
Fig. 10.2 Suggested translocation mechanism for anions of fatty acids (left) and their peroxides
(right) by uncoupling proteins (UCP), ATP/ADP-antiporter (ANT), or aspartate/glutamateantiporter (AGA). It is assumed (left-hand scheme) that UCP1, ANT, and AGA are competent in
electrophoretic efflux of nonoxidized fatty acid anions from the inner to outer leaflet of the inner
mitochondrial membrane. The exported fatty acid anions are protonated by H+ ions from the
mitochondrial intermembrane space and then return to the inner leaflet in their protonated form.
As a result of such a flip-flop, the intermembrane H+ ions are transported to the mitochondrial
matrix (the fatty acid protonophorous cycle). The right-hand scheme illustrates another function
characteristic of UCP2, 3, 4, and 5, namely purification of the inner leaflet from peroxidized fatty
acids. This function is assumed to be characteristic of these minor UCPs. Protonated fatty acid
peroxides cannot return to the inner leaflet since the hydrophilic peroxy group prevents their flipflop. Electrophoretic efflux of fatty acid peroxide may preserve the matrix-located mitochondrial
DNA from being oxidized by very aggressive intermediates of peroxidation of unsaturated fatty
acids (from Goglia and Skulachev 2003)
fatty acids (RCOOH), have very low conductivity for fatty acid anions. Thus, fatty
acids per se are not protonophores. However, in the presence of the RCOO-transferring UCP, the efficiency of fatty acids as uncouplers should dramatically
increase (Fig. 10.2).
The transfer of RCOO- by UCP and ATP/ADP-antiporter, as well as by
aspartate/glutamate-antiporter could proceed due to the interaction of this anion
with positively charged Arg and Lys residues in the lower part of the funnel
formed by these proteins (see previous chapter, Fig. 9.10). The RCOO--anion
could reversibly bind to Arg 234, 235, or 236 of the bottom of the funnel; this
would suffice for these anions to cross the hydrophobic membrane barrier.
The other very important characteristic of fatty acid-mediated uncoupling is that
no special mechanism is needed for the termination of the uncoupling effect in this
case. Uncoupling ceases when the rate of fatty acid influx in mitochondria
becomes lower that the rate of their oxidation. This phenomenon occurs when an
increase in the ambient temperature switches off the cold receptors in the skin;
then the regulatory cascade, which forms fatty acids from triacylglycerides, ceases
to function. When no fatty acids are transported into the mitochondria, their level
is quickly lowered by the b-oxidation system. As a result, the fatty acid-mediated
uncoupling disappears.
Significantly, not only cooling, but at least three other states of the organism,
also related to increased oxygen consumption and heat production,
236
10
D
lH as Energy Source for Heat Production
are accompanied by uncoupling in brown fat tissue. One such state is the birth of a
newborn. Immediately after birth, the young organism suddenly changes the
comfortable isothermal conditions in the mothers body to the hypothermal outer
medium. The small size of a newborn (and thus the high surface/volume ratio)
additionally complicates the problem, for it substantially increases the heat loss.
Brown fat was shown to play the key role in the thermal adaptation of mammal
cubs after their birth. These data explain the fact of this tissue being particularly
well developed in newborns. It also provides some understanding of why the share
of brown fat tissue in body mass decreases during postnatal period parallel to the
increase in body size and decrease in surface/volume ratio.
Another example of brown fat functioning is the arousal of an animal after
hibernation. It was found that brown fat does not undergo postnatal involution in
hibernators adapted to thermoneutral conditions. Cold acclimation increases the
thermogenin concentration in brown fat mitochondria of hibernators, but less
dramatically than in non-hibernators such as rats or rabbits. The uncoupling that
was found to accompany the awakening of a hamster from hibernation appears to
be especially important, because the brown fat warms the blood going to the brain
so that the temperature rises first in the brain (Smith and Horwitz 1969).
Another model is thermogenesis induced by excessive food consumption. Nancy
Rothwell and Michael Stock found that adult rats eating a cafeteria diet of
varied and palatable food increase their usual dietary intake by 80 % (Rothwell
and Stock 1981). But the gain in mass over a 3-week period was only 27 % greater
than that for the control group. They consumed *25 % more oxygen than the
control animals. This increase was abolished by a noradrenaline antagonist, propranolol. The mass of the brown fat after 3 weeks of cafetering was three times
higher than that in the control rats. The UCP concentration in the brown fat
mitochondria also increased. On the other hand, a mutation resulting in obesity of
mice was shown to be accompanied by a decrease in the UCP content in brown fat
mitochondria.
The role of UCP in thermal adaptation has been directly confirmed by
molecular genetic studies. Cannon and Nedergaard obtained a mouse strain with
knockout of the gene for UCP (Nedergaard et al. 1999). These mice were shown to
be much more sensitive to temperature decrease than the wild type.
10.2
237
body mass and containing many mitochondria (e.g. skeletal muscles), should also
participate in heat production in response to cooling. On the other hand, the
thermogenin genes are known to be expressed only in brown fat tissue. Thus, the
heat production mechanism in other tissues of warm-blooded animals may differ
from the mechanism of thermogenesis in brown fat.
One of the thermogenesis mechanisms of skeletal muscles is well known to all
of usit is muscle shivering. It is supported (as well as any other muscle contraction) by hydrolysis of cytoplasmic ATP. The latter is restored by ATP transport
from mitochondria via the ATP/ADP-antiporter. ATP is produced in mitochondria
by H+-ATP-synthase, which utilizes D
lH generated by the respiratory chain (i.e.
in this case thermogenesis is described as respiration ? D
lH ? ATP ? heat).
However, this mechanism of thermal adaptation is also known to be used only
in case of sudden cooling. It seems to be too slow and complicated for effective
long-term acclimation to a decrease in ambient temperature. Moreover, it activates
muscle contraction, i.e., a specific function of this tissue (oxygen consumption in
the case of abrupt cooling is similar to that of heavy muscular work). Thus, the
main principle of thermoregulation, i.e., to make the functions independent of
temperature, remains unresolved. Therefore, it is not surprising that muscle
shivering decreases in the course of cold adaptation. This fact points to the existence of some other mechanism of heat production that is activated in muscles in
the case of the thermoregulatory long-term cooling.
It is interesting that the role of mitochondria in thermogenesis was first shown
for animal skeletal muscles. These experiments studying the in vivo effect of a
cold exposure upon muscle mitochondria were carried out in 1960 by one of this
books authors (V.P.S.) together with Sergei Maslov in the laboratory of Sergei
Severin (Skulachev and Maslov 1960). The experiments were conducted on
pigeons that were shorn beforehand to exclude physical thermoregulation. The
cage with the bird was put into a ventilated refrigerator at - 20 C. The pigeon
survived for 1520 min when exposed to these conditions for the first time. But
birds cooled for the second time the next day were shown to acquire the ability to
survive for hours.
The study of oxidative phosphorylation in mitochondria isolated from the breast
muscle of pigeons cooled for the first time revealed a decrease in the P/O ratio by a
factor of 1.6. The second cold exposure resulted in about a sixfold lowering of the
P/O ratio, i.e., almost complete uncoupling of respiration and phosphorylation.
Further studies of this phenomenon revealed that development of the uncoupling
effect takes *15 min of cold exposure. This was shown not only in birds, but also
in mammals (mice) (Skulachev 1963).
It was also found that the concentration of high-energy phosphates increases
during the first cold exposure and decreases during the repeated one. This fact
showed that muscles were involved in this thermoregulatory response. Measurements of in vivo respiration showed that initially it was stimulated on both the first
and repeated coolings, but then decreased in the former, but not in the latter case. It
looked as if respiration stimulated by the first cold exposure still formed ATP,
being not sufficiently uncoupled. On repeated cooling, a system of fast uncoupling
238
10
D
lH as Energy Source for Heat Production
was activated so that the respiration rate was not limited by formation and
utilization of ATP. If so, one might hope that the injection of an artificial uncoupler
into the animal on the first cooling would save it from sudden death in the cold.
Experiments undertaken by Maslov showed that this was really the case. Injection
of 2, 4-dinitrophenol, which is in fact a poison for animals, significantly prolonged
the survival time for mice on their first cold exposure (Skulachev et al. 1963).
In other experiments, respiration of the intact rat diaphragm muscle was shown
to be stimulated by repeated short-term cold exposure of the animal, whereas the
degree of the respiratory stimulation by 2, 4-dinitrophenol was strongly decreased.
An effect similar to that of cold could be induced by the injection of noradrenaline.
Further studies revealed that the concentration of free fatty acids increased significantly, while that of triglycerides decreased, in the muscle of an animal
repeatedly subjected to short cold exposure (Levachev et al. 1965). Some other
data were obtained indicating that free fatty acids were responsible for coldinduced uncoupling in muscle mitochondria. Addition of delipidated serum
albumin, which binds free fatty acids, increased the P/O ratio in mitochondria from
cold-treated animals. A fraction of free fatty acids isolated from the muscle of
cold-treated animals and added to mitochondria from the untreated control animals
was found to induce uncoupling. Moreover, palmitic acid, when added at a concentration as low as 1 9 10-5 M, caused a significant stimulation of respiration in
muscle mitochondria while in the state of respiratory control.
Thus, fatty acids were shown to serve as thermoregulatory uncoupling mediators in mitochondria of both brown fat and skeletal muscles tissues. However, it
has already been mentioned that muscle mitochondria possess no thermogenin. If
this is the case, what is the mechanism of the uncoupling induced by fatty acids?
Elena Mokhova in our group showed that in muscle mitochondria, uncoupling
induced by low concentrations of fatty acids is specifically abolished by
5 9 10-7 M carboxyatractylate, a specific inhibitor of the ATP/ADP-antiporter
(Andreyev et al. 1988). ADP added with or without oligomycin also decreased the
palmitate-induced stimulation of respiration. On the other hand, fatty acids were
found to inhibit ATP/ADP-antiport. Thus, it was concluded that uncoupling of
muscle mitochondria by fatty acids in their physiological concentration range is
mediated by the ATP/ADP-antiporter. We need to stress once again that UCP is in
many ways similar to the ATP/ADP-antiporter (in terms of its amino acid
sequence and domain structure). The transfer of H+ by UCP is also activated by
fatty acids and inhibited by purine nucleotides (see above). Thus, fatty acids
probably mediate thermoregulatory uncoupling in mitochondria of both muscle
and brown fat tissues, though it is the ATP/ADP-antiporter that is the fatty acid
target in the first case, while in the second one it is thermogenin, a special protein
which might be a modification of an antiporter1 (see Fig. 10.2).
10.2
239
The fact that fatty acids not only increase the permeability of the inner mitochondrial membrane for H+, but also compete with adenine nucleotides for ATP/
ADP-antiporter, should insure the cell from the uncoupling-caused depletion of
extramitochondrial ATP when H+-ATP-synthase starts working in the reverse
(hydrolytic direction). ATP depletion might stop oxidation of fatty acids that serve not
only as a regulator of coupling, but also as the main respiration substrate in the cold. It
has been already mentioned that activation of fatty acids by means of their binding to
CoA at the expense of energy of ATP occurs in the outer mitochondrial membrane.
Studies on mitochondrial uncoupling in tissues other than brown fat have
recently produced quite unexpected results. The mammalian genome was shown to
contain not only the thermogenin (UCP) gene, but also four homologous genes.
The products of these genes were given the names of uncoupling proteins UCP2,
UCP3, UCP4, and UCP5, while thermogenin is now called UCP1 (Adams 2000;
Skulachev 1998, 1999). Moreover, uncoupling proteins were discovered in the
majority of investigated animals (both warm-blooded and cold-blooded) as well as
in many plants (Ledesma et al. 2002; Vercesi et al. 1995). Moreover, some UCPs
probably function also in fungi and protozoa. Such a wide distribution of these
proteins suggests that they play a fundamental role in the metabolism of all
eukaryotes.
The expression of genes of new mammalian uncoupling proteins is tissuespecific. UCP2 is expressed in many tissues, UCP3 in brown fat and skeletal
muscles, while UCP4 and UCP5 are specific for brain. The discovery of a whole
group of UCPs in mammals might suggest that the main function of these proteins
is heat production in tissues other than brown fat. Certain observations seemed to
support this conclusion: exposure of an animal to cold caused the increased
expression of genes UCP2 and UCP3 (quite surprisingly, similar cold induction is
also typical for several plant UCPs). However, it was shown later that the
induction of the uncoupling proteins could be observed not only as a result of
cooling, but also under many other conditions, including, e.g., starvation, when a
decrease in energy storage efficiency could have no positive sense. Thus, the
increased expression of the uncoupling proteins genes probably correlates with the
acceleration of fat metabolism rather than to lowering of temperature.
Moreover, mutant mice with knocked-out genes of different alternative
uncoupling proteins (UCP2UCP5) demonstrate no differences in cold adaptation
when compared to the control mice. Thus, UCP2UCP5 are considered to participate in the cold-stress-induced mitochondrial uncoupling only to a minimal
degree. Fernando Goglia and one of this books authors (V.P.S.) suggested the new
UCPs to be the carriers of fatty acid peroxide anions and hence to be responsible
for the transfer of these dangerous compounds from the inner to the outer halfmembrane layer of the inner mitochondrial membrane (see above Fig. 10.2,
Goglia and Skulachev 2003). This hypothesis might explain numerous data indicating a possible role of the new UCPs in the antioxidant protection of mitochondria, and also the fact that their amount is very small when compared to that
of UCP1 (similar to the quantity of fatty acid peroxides, which is always much
lower than that of intact fatty acids).
240
10
D
lH as Energy Source for Heat Production
10.3
241
Fig. 10.3 (ac)Florescence stages of the thermogenic plant Symplocarpus renifolius, d infrared
picture of the c stage, e, f, g magnified versions of pictures b, c and d. Temperature scale for the
pictures d and g is given in the lower right corner; bar 1 cm (Onda et al. 2008)
Fig. 10.4 Scheme of the mitochondrial respiratory chain of thermogenic plants. Electron transfer
pathways prevalent in thermogenesis are marked with bold arrows
1950). At the same time, coupling mitochondrial enzymes (complexes I, III, and
IV) were shown to provide only 5 % of the overall respiration in these inflorescences. Thus, NDin and NDex shunt the first coupling site, and AOX shunts the
second and the third coupling sites in these mitochondria (Fig. 10.4). This is the
mechanism of direct transformation of respiratory energy to heat. Thus, heat
production mechanisms are fundamentally different in plants and warm-blooded
animals.
References
Adams SH (2000) Uncoupling protein homologs: emerging views of physiological function.
J Nutr 130:711714
Andreyev AYu, Bondareva TO, Dedukhova VI, Mokhova EN, Skulachev VP, Volkov NI (1988)
Carboxyatractylate inhibits the uncoupling effect of free fatty acids. FEBS Lett 226:265269
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lH as Energy Source for Heat Production
Bertsova YV, Popov VN, Bogachev AV (2004) NADH oxidation by mitochondria from the
thermogenic plant Arum orientale. Biochemistry (Moscow) 69:580584
Drahota Z, Alexandre A, Rossi CR, Siliprandi N (1970) Organization and regulation of fatty acid
oxidation in mitochondria of brown adipose tissue. Biochim Biophys Acta 205:491498
Foster DO (1984) Quantitative contribution of brown adipose tissue thermogenesis to overall
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Part IV
Chapter 11
11.1 Regulation of D
l H
The D
lH -generating and D
lH -consuming systems can be regulated by an entire
set of mechanisms usually used by organism to control enzymes. A number of
examples of the regulatory effects have been already described in the previous
chapters. In this chapter, we shall discuss specific systems of D
lH regulation. In
the preceding chapter, we considered those related to the free oxidation in thermoregulation. Below we will show that non- and uncoupled redox processes, often
in combination with energy-coupled ones, are of much greater significance than
heat production.
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11.1
Regulation of D
lH
247
with the outer cell membrane of a leukocyte. This results in a burst of free
respiration mediated by NADPH, flavoproteins, and the autooxidizable, cyanideresistant b-type cytochrome. The latter mediates one-electron reduction of O2 to
superoxide anion O2-, which then reacts with a chloride ion. As a consequence, a
chloride radical is formed, this process being catalyzed by myeloperoxidase. The
chloride radical is a highly toxic compound that kills bacteria (Gabig and Babior
1981). It seems quite important that NADPH is oxidized by flavin on the inner
surface of the leukocyte plasmalemma, while oxygen is reduced on its outer
surface. It is due to such topography that the poisonous product is formed outside
the leukocyte, thus minimizing the risk of the damage to the leukocyte.
The reason why the above-mentioned respiratory systems are not coupled to
D
lH generation seems to be clear enough. Energy coupling must inevitably
complicate the constructive or destructive functions of these systems that are of
vital importance for the organism.
Thus, we have considered the four main functions of cellular oxidations:
(1) conservation of usable energy (D
lH generation); (2) energy dissipation to
form heat; (3) formation of useful substances; and (4) decomposition of harmful
substances. One may think that the first of these functions is performed by coupled
respiration, whereas the three others involve free (non- or uncoupled) respiratory
systems. However, this appears to be an oversimplification.
Sometimes coupled respiration participates in functions (2)(4). Thus, shivering
thermogenesis, the first response of a warm-adapted animal to cold, requires the
formation of D
lH and then ATP to supply actomyosin ATPase with its substrate.
It is also quite clear that the majority of constructive (anabolic) mechanisms utilize
energy of ATP produced by coupled respiration. As to the destructive function,
here ATP is usually not involved, but some important exceptions are well known.
For example, removal of NH3 via the urea cycle consumes ATP.
On the face of it, free respiration, according to the definition, cannot take part in
the energy-conserving function. Paradoxically, this is not always the case.
The initial step of the coupled respiratory chain is known to be not only the
slowest but also the most vulnerable segment of the respiratory chain. A wide
range of hydrophobic xenobiotics arrests the coupled NADH:CoQ-oxidoreductase.
Bypassing the inhibited step by free redox reactions can release respiration, which
now again forms ATP but, of course, at a lower thermodynamic efficiency. This
may be of significance for mitochondria of liver cells specializing in the accumulation and oxidation of hydrophobic xenobiotics by cytochrome P-450.
The shunting of inhibited NADH: CoQ-oxidoreductase can be achieved by a
release of cytochrome c into the intermembrane space or by addition of vitamin K3
(menadione) actuating the dicumarol-sensitive pathway via DT-diaphorase, a watersoluble enzyme oxidizing NAD(P)H by quinones. However, it is not clear whether
such systems demonstrated in vitro are really activated under the conditions of
intoxication by hydrophobic xenobiotics. On the other hand, an artificially induced
shunting by injected menadione had a pronounced medicinal effect on a patient with
a mutation in a respiratory chain enzyme (for details, see Sect. 4.2.4).
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11.1
Regulation of D
lH
249
Fig. 11.1 Shuttle system to oxidize cytosolic NADH by the mitochondrial respiratory chain.
Outer NADH reduces oxaloacetate (OA) by means of reversible cytosolic malate dehydrogenase
(1). The formed malate (Mal) comes into the matrix in exchange for a-ketoglutarate (a-KG) via
dicarboxylate-antiporter (2). In the matrix, malate reduces NAD+ by means of mitochondrial
malate dehydrogenase (3). The resulting oxaloacetate transaminates with glutamate (Glu) to form
aspartate (Asp) and a-ketoglutarate (4). Inner aspartate is exchanged for outer glutamate and H+
via aspartate/(glutamate ? H+)-antiporter in a D
lH -dependent manner (5). Outer aspartate
transaminates with outer a-ketoglutarate, regenerating outer oxaloacetate (6). The D
lH needed
to power reaction (5) is generated by the respiratory chain (7), which oxidizes NADH
250
11
staining in the other cell. There are some indications that mitochondria have their
own K+/H+-antiporter, which is usually in a latent state.
The uniport of Ca2+ into mitochondria may be one of the factors causing
DW ? DpH conversion. However, this process is usually of limited importance
because of a very low (10-710-6 M) Ca2+ level in cytosol. As to the K+ (cytosolic
level *0.1 M), its large-scale uniport must be absent from mitochondria, since K+
distribution down DW would result in the accumulation of a very high concentration of KOH in the matrix space.
The import of K+ seems to be the main mechanism for the DW ? DpH transition in bacteria, which are living at a much lower outer [K+] than mitochondria.
In chloroplasts, D
lH is mainly in the form of DpH due to the high permeability of
the thylakoid membrane for Cl-, K+, and Mg2+.
11.1.4 Relation of D
lH Control to the Main Regulatory
Systems of Eukaryotic Cells
In eukaryotic organisms, supracellular control systems, such as hormones, transmit
their commands via intracellular mediators that are called secondary messengers.
Most common among them are Ca2+, cyclic nucleotides, and products of phosphatidylinositol diphosphate breakdown, i.e., inositol triphosphate and diacylglycerol. Recently, even more attention is also attracted to such a pleiotropic
regulator as H+.
Sometimes metabolites play the role of secondary messengers that regulate
their own metabolism. Previously (see Sect. 10.2.1) we considered such a situation
when discussing the mechanism of fatty acid-mediated thermoregulatory uncoupling. Fatty acids are not only the substrates of oxidation, but also uncouplers of
this oxidation and inhibitors of the ATP/ADP-antiporter. Later, another regulatory
effect of fatty acids was found. This is the release of hexokinase bound to the outer
mitochondrial membrane (Klug et al. 1984). This can facilitate competition of fatty
acids with glucose for ATP in formation of acyl-CoA and glucose-6-phosphate,
respectively. Both these processes can occur on the surface of the outer mitochondrial membrane. Fatty acids were found to increase the negative surface
charge of the membrane, resulting in the decomposition of a complex of hexokinase with porin, a function of which is to be a membrane receptor for hexokinase. Activation of certain enzymes of the Krebs cycle by Ca2+ is also described.
As to the classical secondary messengers, there are numerous publications on
the effects of Ca2+ ions on various mitochondrial functions. The first is temporary
DW ? DpH transition accompanying Ca2+ import. Moreover, Ca2+ activates
mitochondrial phospholipase A2. The latter produces free fatty acids, which cause
uncoupling and other effects described above. Another product, lysophospholipid,
can also influence some parameters of mitochondria.
11.1
Regulation of D
lH
251
Also, cAMP can apparently affect some mitochondrial functions not only via
fatty acid release, but more directly as well. There is a cAMP-binding protein on
the outer surface of the inner membrane of yeast mitochondria. It is a 45 kDa
polypeptide synthesized in cytoplasmic ribosomes. Its affinity to cAMP is very
high (Kd = 10-9 M) (Rdel et al. 1985). One can speculate that it is this protein
that plays the role of a specific receptor responsible for some in vivo effects of
cAMP, namely, an increase in the respiration rate and in the synthesis of subunits
IIII of cytochrome oxidase in mitochondria.
There is no doubt that H+ can also be used as a secondary messenger. For
instance, it is quite obvious that pH regulation is very important for the functioning
of H+-ATPase, the D
lH -generating enzyme of the plant and fungal outer cell
membrane. In fact, H+-ATPase pumps H+ ions from the cytoplasm when cytoplasmic [H+] increases (see Sect. 7.2.3.1). The effect of auxins, plant hormones, is
apparently associated with acidification of the cytoplasm resulting in the stimulation of H+ATPase and the increase in DW across the outer cell membrane
(Brummer and Parish 1983).
11.1.5 Control of D
lH in Bacteria
For many bacteria, parallel pathways of respiratory chain electron transfer are
typical, some of them being coupled to D
lH formation, the others representing
free oxidation systems. Moreover, free and coupled redox reactions are often
consecutively included into the respiratory chain.
An interesting example of a system maintaining high D
lH according to the
self-regulation principle was revealed in motile bacteria. It was shown that artificially induced D
lH changes can cause behavioral responses of bacterial cells
(Miller and Koshland 1977). Thus, the addition of uncouplers or exhaustion of
oxygen, resulting in D
lH decrease, is sensed by bacteria as a repellent, changing
the direction of their movement. On the other hand, an increase in [O2] seems to
produce an attractant signal favorable for linear movement. It was noted that the
effect of O2 upon bacterial behavior (aerotaxis) can be shown only if a change in
[O2] shifts the D
lH level.
It was suggested that bacteria have a device competent in measuring D
lH
across their membrane. By means of this system, D
lH controls the direction of
flagellar rotation: the direction changes when D
lH decreases and remains
unchanged when it increases. As a result, the bacterium moves to a region where it
can maintain higher D
lH (Glagolev 1980). This type of hypothetical mechanism,
called a protometer by one of this books authors (V.P.S.), allows many
favorable and unfavorable effects upon membrane energetics to be integrated.
Functioning of a protometer has been recently directly demonstrated for the taxis
system of H. salinarium (Bibikov et al. 1993) (see Sect. 6.5.3).
A mechanism has been described that coordinates the work of two photosystems in chloroplasts and hence optimizes D
lH and NADPH production.
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If photosystem II works too fast, a redox carrier (presumably PQ) included between
photosystems I and II becomes completely reduced. This activates in some manner
a protein kinase that phosphorylates the protein bearing light-harvesting antenna
chlorophyll localized mainly in stacked thylakoids of grana. Phosphorylation adds
negative charges to the antenna proteins which, due to electric repulsion, diffuse
along the membrane to adjacent stroma membranes where photosystem I is mostly
localized. As a result, photosystem II receives fewer, while photosystem I receives
more photons from the antenna. Activation of photosystem I oxidizes PQH2 and,
hence, inhibits protein kinase. Constitutively, active protein phosphatase dephosphorylates antenna protein and prevents its further outflow from thylakoids to the
stroma lamellas (Staehelin and Arntzen 1983).
11.2
253
254
11
the microplasmodesmata, one might hope that the electric potential difference
generated across the cytoplasmic membrane near, e.g. one of the trichome ends,
can be transmitted along the trichome and utilized in its distal end to carry out
work. Severina found that illumination of the trichome end (about 5 % of the
trichome length) with a small light beam-initiated motility of the trichome.
The measured amplitude and the kinetics of the DW propagation along the trichome were in agreement with the same parameters calculated on the basis of
assumption that the cyanobacterial trichome has the properties of an electric cable.
The conclusion on the cable properties of cyanobacterial trichomes can be
extended to plant tissues in which the cells are connected with plasmodesmata
quite permeable to ions and, in particular, to H+. A study of the possible significance of such a power transmission for plant tissue economy seems to be an
interesting subject for future investigations.
11.2
255
Fig. 11.2 Single mitochondrion in the cell of a flagellate, Polytomella agilis: a Single section
(the picture can be wrongly interpreted as indicating the presence of many small, roundish, or
ellipsoid mitochondria at the cell periphery); b and c front and side views of the model of the single
giant mitochondrion of P. agilis, reconstituted from serial sections (Burton and Moore 1974)
micrographs of the whole cell with the aid of serial thin sections; (2) high-voltage
electron microscopy allowing one to increase the thickness of the studied preparation, and (3) the staining of mitochondria with fluorescent penetrating cations,
thus making it possible to return to studies of mitochondria in the living cell using
the light microscope.
The serial section method was first applied in studies on unicellular eukaryotes.
Here, very large and complicated mitochondrial structures were detected. For
example, in a flagellate, Polytomella agilis, (Burton and Moore 1974) described a
single (!) mitochondrion that looked like a hollow, perforated sphere arranged
immediately below the outer cell membrane so that the cytoplasm and the nucleus
was packed into a mitochondrial string bag (Fig. 11.2).
A single giant mitochondrion was described in some yeast cells, and also
Chlorella and other unicellular algae. Several huge branched mitochondria,
sometimes united into a single mitochondrion, were detected in Chlamydomonas,
Euglena, Trypanosoma, and some fungi. Threadlike, 10-lm-long mitochondria
were found in exocrine cells of the pancreas. A chain of long end-to-end arranged
mitochondria were described in spermatozoa. In liver cells, mitochondria of two
types were foundsmall spherical and the large branched ones. A similar picture
was revealed in ascites tumor cells and in an algal cell, Polytoma papillatum. In
the latter case, 228 consecutive sections were analyzed. It was found that there
were 246 separate small mitochondria, two large and branched ones, and a very
large one forming a perforated, hollow sphere (Smith and Ord 1983).
Muscle tissue seems to be one of the most interesting objects for studying
intracellular power transmission. Very large multinuclear muscle cells (fibers)
have high energy requirements. In a hard-working muscle, gradients of oxygen and
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11
substrates between the periphery and the core of the cell should arise, this effect
limiting the scope of the work performed. The transmission of D
lH from the
muscle cell edge to its core along mitochondrial membranes might solve this
problem. If this were the case, the dimensions of muscle mitochondria should be
particularly large.
In insect flight muscle, slab-like mitochondria of about the same length as the
muscle fiber radius, i.e., 10 lm, have been described.
The first indications of the existence of a mitochondrial system penetrating the
muscle fibers of higher animals were obtained in the 1960s by Bubenzer (1966)
and Gauthier (1969). They studied random sections of rat diaphragm muscle. In
our group, Lora Bakeeva and Yuri Chentsov undertook a systematic investigation
of serial sections of rat diaphragm muscle. It was found that in this tissue the
mitochondrial material is organized into networks transpiercing the I-band regions
of the muscle near the Z-disks (Bakeeva et al. 1977, 1978). These networks are
connected with columns oriented perpendicular to their plane, i.e., parallel to
myofibrils. Moreover, there are branches, arranged parallel to the Z-disks, connecting the networks with mitochondrial clusters at the periphery of the fiber
(Fig. 11.3). Such a system, defined by one of this books authors (V.P.S.) as
mitochondrial reticulum (Reticulum mitochondriale), was found to be characteristic of the diaphragm of adult animals. It is absent from the diaphragm of rat
embryos and newborn rats (Bakeeva et al. 1981).
Sometimes the reticulum forming mitochondrial filaments contain dark partitions built of four membranes, the intermembrane space being filled with osmophilic material. These partitions apparently represent junctions of two branches of
the mitochondrial reticulum This structure, discovered by Bakeeva and her colleagues in diaphragm (Bakeeva et al. 1978), was later studied in detail by the same
group in heart muscle since here mitochondrial junctions were found to be
especially numerous (Bakeeva et al. 1983). It was found that in this tissue that
mitochondria also form a 3D system, but instead of a thin filamentous mitochondrial network found in diaphragm and skeletal muscle, the heart has a multitude of thick, poorly branched organelles. However, they are all coupled to each
other by numerous junctions (Fig. 11.4).
The junction zone was found to be a disk with a diameter of 0.11.0 lm. In
these zones, membranes and intermembrane spaces are of higher density. Two
outer membranes of contacting mitochondria appear to maximally approach each
other in a manner similar to that observed in tight junctions of the outer cell
membranes (Fig. 11.4). Each mitochondrion was shown to be connected with its
neighbors by several such junctions. Mitochondrial contacts could not be detected
in the hearts of 3-day-old rats.
An even more complicated junction structure was found in the zone of the
intercellular gap junction (nexus). Occasionally, one can see that two mitochondria
belonging to two neighboring cells are in close contact with the outer cell membrane. As a result, a junction composed of six membranes is formed (two cellular
and four mitochondrial membranes.
11.2
257
258
11
Filamentous mitochondria. If the studied tissue does not have such a high
electron density as muscle, the real shape and size of mitochondria can be easily
determined by high-voltage electron microscopy. Investigations of this kind
revealed long filamentous mitochondria in diverse cell types.
However, two limitations of this method should be noted. First, it is impossible
to observe mitochondrial junctions, and so one cannot answer the question whether
it is really a single, lengthy mitochondrion or several, end-to-end joined organelles. And second, like any other electron microscopic method, it deals with fixed
material, and thus it is not possible to follow directly the functioning of mitochondria. To overcome this latter limitation, we decided to return to light
microscopy. Here, functional analysis is quite possible, but the great disadvantage
is low resolution that is insufficient to see with certainty thin mitochondrial filaments and networks. We might overcome this limitation when dealing with light
emission instead of light absorption. If the light emission is sufficiently strong, the
light source will be seen in the dark even if it cannot be observed as a lightabsorbing body (that is why the stars in the sky can be seen only at night).
The experiments conducted by Lan Bo Chen and colleagues in the USA (Johnson
et al. 1981) and by Dmitry Zorov in our laboratory (Drachev and Zorov 1986) showed
11.2
259
260
11
required protein synthesis. It is remarkable that only a few mitochondria (no more
than 20 %) were elongated, indicating that some additional function requiring
filamentous mitochondria, besides the small ones, might appear under the changed
conditions.
Another situation was described by (Tanaka et al. 1985). Using serial sections,
they showed that in the yeast Candida albicans at the time of entry into a bud a
few mitochondria fused into a single giant one, which fragmented during mitosis,
returned to a single giant form before cytokinesis, and was then partitioned into
two parts.
The formation of mitochondrial filaments was described as early as 1964 by
Meisel and colleagues in the yeast Endomyces magnusii under hypoxic conditions
(Meisel et al. 1964).
The mitochondrion as an intracellular proton cable. The data discussed in the
preceding section are sufficient for concluding that many types of eukaryotic cells
possess very long filamentous or network-like mitochondrial structures besides,
and sometimes even instead of, the numerous small spherical organelles considered in textbooks as typical mitochondria.
If a mitochondrial filament is a continuum of both inner and outer membranes,
D
lH will be transmitted throughout the entire length of the filament. Yet, if the
mitochondrial filament is an assembly of many small contacting end-to-end
mitochondria or mitoplasts, two possibilities should be considered: (i) the filament
is similar to a cyanobacterial trichome where the constituents (individual cells) are
connected to each other by junctions of high electrical conductance (this chapter,
see Sect. 11.2.3); (ii) there is no electrical contact of adjacent mitochondria in the
filament.
In the preceding section, we described the intermitochondrial junctions that
clearly represent specific structures responsible for contact of adjacent mitochondria. It remained obscure, however, whether the electric resistance of the
junctions is as high as in other regions of the inner mitochondrial membrane or
alternatively is it as low as in cyanobacterial microplasmodesmata or gap junctions
of two animal outer cell membranes. The latter version is identical to the mitochondrial continuum in the sense of D
lH transmission. The former possibility
requires an exchange of energy equivalents other than D
lH between neighboring
mitochondria to be postulated. These equivalents could be ATP shuttling through
mitochondrial junctions.
An obvious prediction of the hypothesis considering a filamentous mitochondrion as cable is that the whole filament must be de-energized when any of its parts
become leaky. This prediction was verified in the group of one of this books
author (V.P.S.). Vladimir Drachev (Fig. 11.6) constructed a special device combining a laser and a fluorescence microscope. Using this device, Dmitry Zorov
succeeded in illuminating a single mitochondrial filament in a human fibroblast
cell stained with ethylrhodamine. A very narrow laser beam (the diameter of the
light spot was comparable with the thickness of the mitochondrial filaments) was
used to cause local damage to the filament. The laser treatment resulted in the
disappearance of the rhodamine fluorescence in the entire 40-lm-long
11.2
Fig. 11.6
261
Vladimir Drachev
262
11
Fig. 11.7 Illumination by a narrow laser beam of a small part of the mitochondrial filament
results in DW collapse over the entire filament length. A cell of the primary culture of human
fibroblasts was stained with ethylrhodamine, a Before and be after 100-ms laser treatment. The
laser light spot was commensurate with the mitochondrial filament thickness. a and b Fluorescent
microscopy. c Phase-contrast microscopy. d The top view on the model of mitochondria in the
fibroblast, reconstituted with the use of the serial section technique. Arrows show the place
illuminated by the laser. e Electron microscopy; a part of the laser-treated mitochondrion is seen
(Drachev and Zorov 1986; Amchenkova et al. 1988)
11.2
Fig. 11.8
muscles
263
Scheme of lateral D
lH transmission along a mitochondrial filament of skeletal
ions. In fact, the filaments might be a route to any substance that is concentrated in
a mitochondrial compartment, including the membranes. For instance, if Ca2+ ions
are transported from the intercellular space to cytosol at the cell periphery, they
can be accumulated in the matrix space of mitochondrial filaments by means of
DW-dependent Ca2+-uniporter, diffuse along the filament as in a tube, and be
released to cytosol in the cell core if the mitochondrial Ca2+/H+-antiporter is
activated in this region of the filament. The same logic can be applied to transport
of any other solute that can be reversibly accumulated in the matrix.
Lateral transport of lipid-soluble compounds can be another variation of this
theme. It is generally believed that transports occurring in a membrane must be
slower than in the water phase of the cell since the viscosity of the membrane is
much higher than that of water, and in this respect the water route should have an
advantage over the membrane route for any amphiphilic compound.
However, one must take into account that the cytosol and the mitochondrial
matrix are not aqueous solutions of low molecular mass compounds, but rather
colloid solutions that are crossed by insoluble structures, namely, by cytoskeleton
and membranes. It is not surprising therefore that a spin-labeled synthetic compound of low molecular mass, of which the partition coefficient in a lipid/water
system is close to 1, was shown to move preferentially in the lipid phase of various
types of cells (Keith and Snipes 1974). Such preference must be even more pronounced for substances for which the lipid/water distribution coefficient is higher
than 1, such as free fatty acids and their carnitine and CoA esters. For the latter, its
role in the lateral transport of fatty acyls was postulated by Manfred Sumper and
Hermann Trauble (Sumper and Truble 1973). Fatty acyl carnitine seems adapted
to this function even better since its hydrophilic moiety is much smaller than in
fatty acyl-CoA.
264
11
11.2
265
Fig. 11.9 Electron transport from the end of one cytochrome b5-bearing membrane filament to
the beginning of another filament; arrows indicate electron transport. It is assumed that
3+
cytochromes b5 in the left and in the right filaments are reduced (b2+
5 ) and oxidized (b5 ),
respectively
a quite measurable level. This preparation completely lost the ability to catalyze
the intermembrane electron transport.
An interesting finding was published by Susan Greenhut and Mark Roseman
(Greenhut and Roseman 1985). When studying the amount of cytochrome b5 in
proteoliposomes differing in diameter by a factor of about 1.5 (21 and 35 nm), they
found that the cytochrome preferred the smaller vesicles to the larger ones by a
factor of at least 20. This means that the formation of highly curved regions of
membranes in the ends, e.g., of mitochondrial filaments, may cause accumulation
of cytochrome b5.
Figure 11.9 illustrates the possible mechanism of electron transfer across the
gap between the ends of two adjacent mitochondrial filaments. It has been proposed that the molecules of cytochrome b5 diffuse on the membrane plane along
the filaments by means of Brownian motion, scanning this membrane and
searching for oxidants. In a region where the distance between two adjacent
membranes does not exceed that of double the diameter of the cytochrome b5
hydrophilic head, cytochrome b5 of the more reduced filament loses an electron to
cytochrome b5 of the more oxidized one. This reaction should be facilitated by the
accumulation of cytochrome b5 at the ends of the membrane filaments, which
might be an example of a taxis of membrane enzymes to their substrates
(Skulachev 1980, 1988).
11.3 Buffering of D
lH
11.3.1 Na+/K+ Gradients as a D
lH Buffer in Bacteria
To perform the role of a convertible energy currency, equivalents of D
lH must be
present in an amount sufficiently large to buffer the rate fluctuations of the D
lH producing and D
lH -consuming processes (Mitchell 1977; Skulachev 1977).
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Taking into account the electrical capacitance of the bacterial membrane, one
can calculate how many H+ ions should be exported from the bacterium to form a
D
lH of about 250 mV (the higher limit of the D
lH value that can be maintained
under energized conditions). Assuming that the capacitance is about 1 lF per cm2,
the number of exported H+ ions is as low as 1 lmol H+ ions per gram of protein,
which is comparable to the amount of enzyme in the membrane (Mitchell 1968). To
store membrane-linked energy in substrate (rather than in catalytic) quantities,
one must discharge the membrane by a transmembrane flux of ion(s) other than H+.
A flow of penetrating ions across the membrane discharges DW and, hence, allows
an additional portion of H+ ions to be exported from the bacterium by D
lH generators until DpH reaches the value equivalent to 250 mV. Now DpH, rather than
DW, appears to be a factor limiting the activity of D
lH generators. The pH buffering capacity of the cytoplasm is by about two orders of magnitude higher than the
electrical capacity of the cytoplasmic membrane, so that the amount of energy
stored in DpH appears to be much higher than that in DW (Skulachev 1978, 1979).
A further increase in the amount of the stored membrane-linked energy can be
achieved if the formed DpH is utilized, for example, by a cation/H+-antiporter to
substitute Dp(cation) for DpH. The involvement of this antiporter exchanging, e.g.
inner Na+ for outer H+, is equivalent to an increase in the concentration of pH
buffer, as recognized already in 1968 by Mitchell (1968). Unfortunately, Mitchell
ignored the role of the other monovalent cation, K+. Accumulation of K+ down DW
was, according to him, an unfavorable but inevitable side effect.
In 1978, one of this books authors (V.P.S.) suggested that influx of K+ is used
by the bacterial cell to induce DW ? (DpH, DpK) transition (Skulachev 1978).
The formed DpK was postulated to buffer the DW component of D
lH . As to DpH,
it is utilized by a Na+/H+-antiporter so that DpNa buffers the DpH component of
D
lH . According to this concept, K+ is accumulated in, and Na+ is exported from,
the bacterial cell when there is an excess of energy. Energy deficiency results in
some lowering of DW and DpH. This causes K+ efflux and Na+ influx, preventing
immediate dissipation of DW and DpH, respectively (Fig. 11.10).
The hypothesis of a D
lH buffer made it possible to explain a number of
observations connected to bacterial energetics. It was found, in particular, that K+
can be electrophoretically accumulated in bacterial cells. Na+/H+-antiport was
described in 1974 by West and Mitchell in E. coli (1974). Since then, Na+/H+antiport has been found in many species of bacteria (Krulwich 1983).
Cooperation of the DW-driven K+ influx and DpH-driven Na+ efflux seems to
meet the requirements for the D
lH buffer. Na+ is the most common cation of the
environment. Its concentration in seawater is as high as 0.5 M. Thus, a cell
pumping out Na+ ions can easily create a large DpNa. [K+]out is usually much
lower than [Na+]out, so a large DpK can be obtained via K+-import. On the other
hand, [K+]out is not so low as to hinder the search for this cation in the medium. In
fact, K+ ranks second among cations in its concentration in seawater. Substitution
of K+ for Na+ and vice versa inside the cell would hardly exert a dramatic
influence upon metabolism and the cell structure owing to the similarity of many
11.3
Buffering of D
lH
267
properties of these two monovalent cations and also because they are not directly
involved in enzymatic processes.
Studies carried out in our laboratory by Brown et al. (1983) confirmed the
hypothesis that bacteria really create Na+/K+ gradients when there is an energy
surplus, and they utilize these gradients to perform useful work when external
energy sources are exhausted. The following observations seem to be noteworthy.
(1) The Na+ and K+ gradients formed when external energy sources were available can sustain such a D
lH -linked function as the motility of halophilic
Halobacterium salinarium, marine Vibrio harveyi, and enterobacterium E. coli
for some time after the depletion of these energy sources. In the freshwater
cyanobacterium Phormidium uncinatum, motility could be supported only by
the K+ gradient, DpNa being ineffective.
(2) The Na+ and K+ gradients stabilize the ATP level when other energy sources
are absent.
(3) The capacitance of the Na+/K+ energy buffer is directly proportional to the
ambient salt concentration, decreasing in the environment for bacteria in the
series H. salinarium [ V. harveyi [ E. coli [ Ph. uncinatum. In H. salinarium preincubated in the light, Na+ and K+ gradients are still able to support
measurable motility for more than 8 h after the cessation of illumination and
exhaustion of O2. This means that halobacteria can invest a very large amount
of energy into the Na+/K+ gradients during the light hours when solar energy is
available, to provide it throughout the night.
It should be emphasized that cooperation of K+-uniport and electroneutral Na+/
H -antiport is the simplest, but not the only possible mechanism for D
lH buffering by cation gradients. For instance, electrogenic Na+/nH+-antiport apparently
+
268
11
11.3.2 Other D
lH -Buffering Systems
A biological membrane usually divides a volume into two unequal parts. Therefore,
H+ pumping across the membrane changes the pH mainly (or even exclusively) in
the smaller compartment. If there are no enzymes in this compartment, the D
lH buffering mechanism can be simplified such that its Na+ part is eliminated. The
most demonstrative example of this phenomenon is chloroplast thylakoids.
Here, H+ ions are released into a narrow intrathylakoid cavity, the size of which
is of the same order of magnitude with that of the membrane. This cavity is
practically empty of enzymes, and thus there is no risk of enzyme inactivation by
strong acidification. As mentioned previously (see Sect. 2.3.4), D
lH buffering in
thylakoids is achieved via DW ? DpH conversion accompanied by the transport
of K+, Mg2+, and Cl- across the thylakoid membrane, while Na+/H+-antiport is
absent. Since the intrathylakoid space is very small in comparison to the chloroplast stroma, DpH (up to three units) is formed almost exclusively due to the
increase in intrathylakoid [H+].
Some D
lH buffering is apparently necessary for chloroplasts because fluctuations in light intensity always occur under natural conditions. This is probably
why chloroplasts convert DW to DpH. A similar problem exists in bacterial
chromatophores. In Rhodospirillum rubrum it can be solved by the involvement of
H+-PPi synthase. This enzyme utilizes D
lH to synthesize PPi from Pi. No rapid
References
269
References
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of NADH oxidation in mitochondria at decreased pH. Biokhimiia 46:19451956 (in Russian)
Amchenkova AA, Bakeeva LE, Chentsov YS, Skulachev VP, Zorov DB (1988) Coupling
membranes as energy-transmitting cables. I. Filamentous mitochondria in fibroblasts and
mitochondrial clusters in cardiomyocytes. J Cell Biol 107:481495
Appleby ML, Morton RK (1959) Lactic dehydrogenase and cytochrome b2 of bakers yeast:
purification and crystallization. Biochem J 71:492499
Archakov AI, Karyakin AV, Skulachev VP (1975) Intermembrane electron transport in the
absence of added water-soluble carriers. Biochim Biophys Acta 408:93100
Bakeeva LE, Chentsov YS, Skulachev VP (1978) Mitochondrial framework (Reticulum
mitochondriale) in rat diaphragm muscle. Biochim Biophys Acta 501:349369
Bakeeva LE, Chentsov YS, Skulachev VP (1981) Ontogenesis of mitochondrial reticulum in rat
diaphragm muscle. Eur J Cell Biol 25:175181
Bakeeva LE, Chentsov YS, Skulachev VP (1983) Intermitochondrial contacts in myocardiocytes.
J Mol Cell Cardiol 15:413420
Bakeeva LE, Skulachev VP, Chentsov YS (1977) [Mitochondrial reticulum: organization and
possible functions of a novel intracellular structure in the muscle tissue.] Vestn Mosk Univ
Ser. Biol 3:2328 (in Russian)
Bakker EP, Harold FM (1980) Energy coupling to potassium transport in Streptococcus faecalis.
Interplay of ATP and the protonmotive force. J Biol Chem 255:433440
Bibikov SI, Grishanin RN, Kaulen AD, Marwan W, Oesterhelt D, Skulachev VP (1993)
Bacteriorhodopsin is involved in halobacterial photoreception. Proc Natl Acad Sci U S A
90:94469450
270
11
References
271
Part V
Chapter 12
D
lNa Generators
12:1
where n appears to be two (Dimroth et al. 2001).
This process could be shown in intact cells, in inside-out subcellular vesicles,
and in proteoliposomes. The biotin-containing enzyme is located in the cytoplasmic membrane of K. aerogenes. The reaction sequence includes two stages:
(1) transfer of the carboxyl residue from oxaloacetate to the biotin prosthetic
group; (2) release of free CO2 from carboxylated biotin with the regeneration of
the initial form of the enzyme. It is the second step that requires Na+ (Dimroth
1982). The decarboxylation-dependent Na+ uptake by inside-out subcellular vesicles resulted in a positive charging of the intravesicular space, this indicating that
the Na+ transport is electrogenic. The DW and DpNa were shown to be about 65
and 50 mV, respectively, the total D
lNa being about 115 mV.
Oxaloacetate decarboxylase from K. aerogenes is composed of three different
subunits: a, b, and c (65, 34, and 12 kDa, respectively). The a-subunit is a
peripheral membrane protein, while the two others are integral membrane proteins
that can be released only by detergent treatment. Biotin is covalently bound to a
conservative lysine residue of the a-subunit (Dimroth et al. 2001).
The functioning of the Na+-translocating decarboxylase makes it possible for
K. aerogenes to ferment such an unusual for anaerobic growth substrate as citrate
275
276
12
D
lNa Generators
Fig. 12.1 Scheme of citrate fermentation in K. aerogenes and K. pneumonia (for explanations,
see text)
(Fig. 12.1). Citrate is first imported into the cell via a Na+-motive citrate carrier
(CitS). It is then decomposed by citrate lyase (CL) to oxaloacetate and acetate.
Oxaloacetate is decarboxylated by the respective decarboxylase, thus forming
pyruvate. This process is coupled to Na+ export, i.e., the energy of this reaction is
stored in the form of D
lNa . Pyruvate formate lyase (PFL) catalyzes transformation of pyruvate to acetyl-CoA and the release of formate. The energy of the
acetyl-CoA thioester bond can be used to form ATP. This process is catalyzed by
phosphotransacetylase (PTA) and acetate kinase (AK). Formate, being toxic for
the cell, is decomposed to CO2 and H2 by formate hydrogen-lyase (FHL), while
electrons are transferred from H2 to NAD(P)+ by hydrogenase. Thus, substrate
phosphorylation produces 1 mole of ATP per mole of substrate in the course of this
fermentation. A certain amount of energy is additionally stored in the form of
D
lNa which can be used for the transport of citrate and other metabolites. The DW
component of D
lNa might be utilized by H+-ATP synthase for some additional
ATP synthesis.
Further research showed that K. aerogenes is not the only microorganism having
a Na+-translocating decarboxylase. So far, four enzymes of this class dealing with
different substrates have been described in various bacteria (Buckel 2001).
A Na+-translocating oxaloacetate decarboxylase was discovered in enterobacteria K. aerogenes, K. pneumoniae, and Salmonella typhimurium.
Microorganisms, such as Acidaminococcus fermentans, Peptococcus aerogenes, Clostridium symbiosum, and Fusobacterium nucleatum have Na+-translocating glutaconyl-CoA decarboxylase, which decarboxylates glutaconyl-CoA (an
intermediate of the fermentation of glutamate to acetate and butyrate) to form
crotonyl-CoA, the reaction being coupled to Na+ export.
In the case of strictly anaerobic Veillonella alcalescens converting lactate into
acetate and propionate and in Propionigenium modestum, which utilizes succinate
and forms propionate, decarboxylation of methylmalonyl-CoA to propionyl-CoA
is also a Na+-motive process. Before decarboxylation, succinate is converted to
12.1
Na+-Motive Decarboxylases
277
278
12
D
lNa Generators
Table 12.1 Properties of Na+-NQR subunits exemplified by the enzyme from V. harveyi, and
their probable functions
Molecular Total number of Prosthetic groups and
Possible function
Na+NQR
mass
transmembrane
cofactors
subunit (kDa)
a-helices
NqrA
48.4
NqrB
45.4
NqrC
27.5
NqrD
22.7
NqrE
21.5
NqrF
45.2
Ubiquinone-8
Quinone-reductase center
of the enzyme
Covalently bound FMN
Electron transfer. Possibly
residue, noncovalently
involved in quinone
bound riboflavin
binding
and/or Na+
translocation
Covalently bound FMN
Electron transfer
residue
Contains two conservative Possibly involved
cysteine residues
in Na+ translocation
Contains two conservative Possibly involved in Na+
translocation
cysteine residues
Noncovalently bound FAD Binding site for NADH,
and [2Fe2S] cluster
responsible for NADH
dehydrogenase activity
of the enzyme
12.2
Na+-Translocating NADH:Quinone-Oxidoreductase
279
NADH H
in CoQ 2 Nain ! NAD CoQH2 2 Naout :
12:2
As translocation of sodium ions is a part of the reaction catalyzed by the Na+NQR, its rate depends on the concentration of these ions. Indeed, NADH oxidation
by CoQ is practically absent in media depleted of Na+. At the physiological values
of ionic strength, the Km for sodium ions are around millimolar values for Na+-
This type of covalent bond between flavin and protein is quite unique; it has not been found in
any studied protein besides the Na+-NQR.
280
12
D
lNa Generators
NQR from different Vibrio species. The Na+-NQR has absolute specificity for Na+.
As to other cations, only lithium was found to activate reaction (12.2). The Na+NQR cannot translocate protons even in the absence of sodium ions.
Translocation of sodium ions by the enzyme is accompanied by the generation
of DW. The protons released from NADH upon its oxidation come to the cytoplasm, and the protons necessary for ubiquinol formation are also taken up from
the cytoplasm. Thus, the Na+-NQR functions as a primary electrogenic sodium
pump, and not as a Na+/H+-antiporter (Zhou et al. 1999).
Na+-NQR has a pH optimum between 8.0 and 9.0. It is specifically inhibited by
very low concentrations of either Ag+ ions or 2-heptyl-4-oxy-quinoline-N-oxide
(HQNO). The antibiotic korormicin is also inhibitory (Hayashi et al. 2001).
The mechanism of sodium potential generation by the Na+-NQR remains
obscure. However, the stage of catalytic cycle that is specifically activated by
sodium ions has been established. As found by an author of this book (A.V.B.) and
Verkhovsky, this is electron transfer from FAD to covalently bound FMN residues
(Bogachev et al. 2002; Verkhovsky and Bogachev 2010).
12:3
DG00 30kJ=mol:
This reaction is catalyzed by the membrane Na+-translocating methyltransferase
complex, and the free energy of this exergonic reaction is stored in the form of
D
lNa . The methyltransferase complex consists of eight different subunits (MtrA-H)
encoded by eight genes of the mtrA-H operon. This protein contains 5-oxybenzimidazolyl cobalamin (cobalamin is a cobalt-containing cofactor, see Appendix 2) as a
12.3
281
prosthetic group bound to MtrA subunit. During the methyl transferase reaction, the
methyl group is first transferred from CH3 H4MPT to cobalamin:
CH3 H4 MPT cobalaminCo ! H4 MPT CH3 cobalamin Co3 :
12:4
The methyl group is then transferred from cobalamin to CoM:
CH3 cobalamin Co3 CoM ! cobalaminCo CH3 CoM 12:5
It is reaction 12.5 that is specifically activated by Na+ and is coupled to
transmembrane electrogenic export of these ions from the cell. Two sodium ions
are assumed to be transported across the membrane per each methyl group
transferred. The mechanism of sodium potential generation by the methyltransferase complex has not yet been elucidated.
12:6
DG0 16kJ=mol
It is not clear yet which carrier of reducing equivalents functions as the electron
acceptor for this reaction. Formylmethanofuran dehydrogenase is an electrogenic
sodium pump; it carries out a transmembrane movement of 23 sodium ions per
CO2 molecule formed (Kaesler and Schnheit 1989).
Formyl-MFR oxidation occurs on conversion of formate or methanol to
methane and CO2:
12:7
12:8
Methanofuran (MFR) is a unique coenzyme of methanogenic archaea that plays the key role in
the reactions of CO2 fixation and reduction, and also in the reactions of oxidation of organic
molecules, the latter being accompanied by CO2 formation. The redox potential of the formylMFR/MFR ? CO2 couple is about -530 mV.
282
12
D
lNa Generators
12.5 Secondary D
lNa Generators: Na+-Motive ATPases
and Na+-Pyrophosphatase
12.5.1 Bacterial Na+-ATPases
In 1982, Heefner and Harold (Heefner and Harold 1982) presented convincing
evidence for a Na+-motive ATPase in the anaerobic bacterium Enterococcus hirae
(at that time this microorganism was called Streptococcus faecalis). It was found that
inside-out subcellular vesicles show a sodium-activated ATPase activity and that
radioactive Na+ is accumulated in the vesicle interior in an ATP-dependent manner.
It was later discovered that mainly a canonical H+-translocating ATPase of
FoF1 type functions in E. hirae when this bacterium grows at neutral pH and low
[Na+], i.e., under optimal conditions. However, increase in [Na+] in the growth
medium, alkalinization of the medium, or growth in the presence of protonophores
result in the appearance of an alternative enzyme, Na+-translocating ATPase of
V0V1 type, which prevails under these conditions (Murata et al. 2001).
This enzyme (as well as other V-type ATPases) was found to be resistant to
azide and vanadate but sensitive to nitrate and N-ethylmaleimide. It is specifically
activated by sodium ions (with two Km values of 20 lM and 4 mM); out of all
other cations, only lithium ions have a similar effect (with Km of 60 lM and
3.5 mM).3 Similar to Na+-motive NADH:quinone-oxidoreductase from V. alginolyticus, Na+-ATPase from E. hirae is mainly active under alkaline conditions
(pH 8.09.0). As discussed in Sect. 7.2.2, hydrolysis of one ATP molecule by Na+translocating ATPase from E. hirae should be coupled to transmembrane transport
of 3.3 sodium ions (Murata et al. 2005).
One may speculate that the biological function of E. hirae Na+-motive ATPase
is to energize the membrane of this glycolyzing bacterium when it is impossible to
employ the usual chain of events leading to metabolite accumulation and fulfillment of other types of membrane-linked work (see Eq. 12.9):
glucose ! ATP ! D
lH ! membrane linked work:
3
12:9
It is interesting that in contrast to Na+, increase in [Li+] in the growth medium does not cause
increased expression of the E. hirae Na+-motive ATPase genes (even though Li+ can be
transported by the enzyme).
12.5
Secondary D
lNa Generators: Na+-Motive ATPases and Na+-Pyrophosphatase
283
In the absence of D
lH , an alternative energy-transducing pathway seems to be
induced:
glucose ! ATP ! D
lNa ! membrane - linked work;
12:10
where the second and the third steps are catalyzed by the Na+-motive ATPase and
the Na+-dependent transporters, respectively.
Bacterial genome studies showed the Na+-translocating ATPases (Na+-ATP
synthases) to be present in a substantial number of bacteria and archaea (Mulkidjanian et al. 2008).
284
12
D
lNa Generators
12.5
Secondary D
lNa Generators: Na+-Motive ATPases and Na+-Pyrophosphatase
285
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Chapter 13
Utilization of D
lNa Produced by Primary
D
lNa Generators
287
288
13 Utilization of D
lNa Produced by Primary D
lNa Generators
13:1
so that the pHin values appear to be equal to 7.4 and 7.8 at pHout 5.5 and 9.0,
respectively.
When pHout decreases, acidification of the cytoplasm can be prevented by K+
import discharging DW that is produced by the D
lH generator. As a result, DW is
converted to DpH, the cytoplasm becoming more alkaline than the outer medium
(Fig. 13.1a).
At neutral pHout, the only problem is how to return to the cytoplasm those H+
ions that are pumped from the cell by D
lH generators. The problem can be solved
by an electroneutral Na+/H+-antiporter (Fig. 13.1b).
The electroneutral Na+/H+-antiporter is effective in the maintenance of pH
homeostasis only if pHout B pHin. Other systems are necessary to acidify the
cytoplasm when pHout is higher than pHin. Bacteria confront this problem when the
growth medium becomes alkaline. Now, there is no reason for the electroneutral
Na+/H+-antiporter to carry H+ from the outside to the interior of the cell since both
Na+ and H+ gradients are in an unfavorable direction. One can overcome this
difficulty, assuming that at alkaline pHout the Na+/H+-antiporter is electrogenic,
transporting more than one H+ per Na+ (e.g. exchanging one Na+ ion for two H+
ions) and the DW formed by D
lH generators is so large that it can compensate for
the unfavorable DpH and DpNa. In such a case, cooperation of a D
lH generator
and a Na+/2H+-antiporter will maintain pHin \ pHout (see Fig. 13.1c).
13.1
289
Fig. 13.1 Possible mechanisms for pH homeostasis in bacterial cytoplasm. a pHout \ pHin; a
D
lH generator pumps H+ from the cell and forms DW (reaction 1). This DW is discharged by the
K+ influx so that DW ? DpH transition occurs, the cell interior being more alkaline (reaction 2);
lH generators (reaction 1) is compensated
b pHout & pHin; the H+ loss due to the activity of D
by H out influx in exchange for Na+in (reaction 2); c and d pHout [ pHin; c the H+-pump exports
H+ and forms DW (reaction 1). To discharge DW, Na+/2H+-antiporter exchanges one Na in for
two H out (reaction 2); d a primary Na+-pump charges the membrane, exporting Na+ (reaction 1).
Uniport of H+ ions into the cell discharges DW and acidifies the cytoplasm (reaction 2)
290
lNa Generators
13 Utilization of D
lNa Produced by Primary D
13.2
Mechanical Work
291
proton motors. Moreover, the sodium motor stator contains also two additional
proteinsMotX and MotYthat are absent from H+-motive flagella (Yorimitsu
et al. 1999). It is noteworthy that the replacement of the Vibrio MotA, MotB,
MotX, and MotY proteins with the proteins MotA and MotB from E. coli changes
the motor specificity: in this case it uses the energy of D
lH (and not D
lNa ) for its
rotation (Asai et al. 2003).
It was recently demonstrated that when growing in the liquid medium,
V. alginolyticus moves due to the functioning of the above-described single polar
sodium flagellum. However, growth in very viscous medium causes V. alginolyticus to express additional smaller flagella that are located laterally on the whole
surface of the bacterium. It is the energy of D
lH (and not D
lNa ) that supports the
rotation of these lateral flagella. Thus, this bacterium can use both D
lNa and D
lH
for performing mechanical work, the choice being dependent on the growth
conditions (Atsumi et al. 1992). An interesting example of energization of bacterial motility was described for the marine microorganism Shewanella oneidensis
MR-1. It was shown that two different stator systems (H+- and Na+-dependent) can
drive a single polar flagellum of this microorganism (Paulick et al. 2009).
Motility supported by D
lNa was also described in other marine vibrios, in
particular, in Vibrio cholerae. It is common knowledge that V. cholerae produces a
toxin causing salt export from tissues to the intestinal lumen. This salt may be
necessary for V. cholerae to support its Na+ energetics.
Motility supported by D
lNa was also demonstrated in alkalophilic Bacilli, for
instance in Bacillus sp. YN-1 (Hirota and Imae 1983), Bac. pseudofirmus FTU
(Bogachev et al. 1993), and Bac. firmus (Kitada et al. 1982). In the Bacillus sp. YN1 studied in detail, Na+ requirement for motility and partial resistance of motility
to protonophores were shown. It was also found that DpNa and DW, generated
enzymatically, are equivalent in supporting motility of this microorganism.
13.3.1 D
lNa -Driven ATP Synthesis in Anaerobic Bacteria
Some evidence that D
lNa ? ATP energy transduction really occurs in some living
organisms was obtained by Peter Dimroth and colleagues (Hilpert and Dimroth
292
lNa Generators
13 Utilization of D
lNa Produced by Primary D
Fig. 13.3 Possible mechanism of energy supply in anaerobic bacterium P. modestum. Succinate
enters the cell where it is converted to succinyl-CoA by transacylation with propionyl-CoA. From
succinyl-CoA, methylmalonyl-CoA is formed. The latter is decarboxylated to propionyl-CoA and
CO2, this process being coupled to Na+-pumping. Exported Na+ ions return to the cytoplasm via
Na+-ATP synthase, which produces ATP from ADP and inorganic phosphate
13:2
This fact explains why Schink and Pfenig (1982) called this microbe modestum, i.e., modest
with respect to its energy requirement.
13.3
Chemical Work
293
ions across the membrane. Interestingly, the same enzyme can also transport
protons when pH is low and Na+ ions are absent from the medium.
Besides sodium ions and protons, ATPase from P. modestum is also able to
transport Li+, but its affinity for this ion is substantially lower. Ion specificity of
Na+-ATPase is determined by the Fo factor. For instance, the chimeric enzyme
composed of the Fo factor from P. modestum and the F1 factor from E. coli
demonstrates the properties of the typical Na+-pump (Gerike et al. 1995).
Besides P. modestum, Na+-ATP-synthases have been described in such bacteria
as Acetobacterium woodii (Mller et al. 2001) and Ilyobacter tartaricus (Neumann
et al. 1998). These enzymes seem to be rather widespread among microorganisms,
in particular in methanogenic archaea (Mulkidjanian et al. 2008).
13.3.2 D
lNa Consumers Performing Chemical Work
in Methanogenic Archaea
It has been already described that two enzymes of methanogenic archaea, i.e.,
methyltransferase complex and formylmethanofuran dehydrogenase, function as
primary sodium pumps (Gottschalk and Thauer 2001; Poorter et al. 2003). However, under certain conditions these enzymes can catalyze the reverse reactions,
i.e., they can utilize D
lNa for performing chemical work (e.g. for methyl group
transfer from methyl-CoM to H4MPT and for reduction of CO2 to formyl-MFR).
In the case of a methanogenic process such as:
CH3 COOH ! CH4 CO2
13:3
CO2 4 H2 ! CH4 2 H2 O
13:4
H2 CO 3 H2 ! CH4 2 H2 O,
13:5
13:6
the same enzyme catalyzes the reverse reaction and acts as the D
lNa consumer.
Na+-translocating formylmethanofuran dehydrogenase functions as a sodium
potential generator when participating in conversion of formate or methanol to
methane and carbon dioxide:
4 HCOOH ! CH4 3 CO2 2 H2 O
13:7
13:8
294
lNa Generators
13 Utilization of D
lNa Produced by Primary D
13:9
References
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driven by different ion-motive forces. Nature 355:182184
Bassilana M, Damiano-Forano E, Leblanc G (1985) Effect of membrane potential on the kinetic
parameters of the Na+ or H+ melibiose symport in Escherichia coli membrane vesicles.
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Bogachev AV, Murtasina RA, Shestopalov AI, Skulachev VP (1993) The role of proton and
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de Poorter LMI, Geerts WG, Theuvenet APR, Keltjens JT (2003) Bioenergetics of the formylmethanofuran dehydrogenase and heterodisulfide reductase reactions in Methanothermobacter thermautotrophicus. Eur J Biochem 270:6675
Dibrov PA, Kostyrko VA, Lazarova RL, Skulachev VP, Smirnova IA (1986) The sodium cycle.
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Hilpert W, Dimroth P (1984) Reconstitution of Na+ transport from purified methylmalonyl-CoA
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Kluge C, Laubinger W, Dimroth P (1992) The Na+-translocating ATPase of Propionigenium
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primacy of sodium bioenergetics. Biol Direct 3:13
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Part VI
Chapter 15
In this part of the book, we will apply the data discussed in earlier chapters on
biological membrane energetics to the solution of a quite practical problem which
is of great importance, namely an attempt to retard or perhaps even eliminate
human aging.
The main points of our concept are as follows:
(1) Aging of living organisms is the final stage of the program of ontogenesis.
(2) Aging is one of the mechanisms accelerating biological evolution. Minor
favorable traits that are not essential for a strong young organism can be of
vital importance (and hence become an object of natural selection) for
organisms weakened by aging.
(3) Humans have already practically ceased to be objects of biological evolution.
We do not adapt to the environmentwe rather change the environment
according to our purposes. Thus, in the case of human beings, aging (as well as
any other evolutionary mechanism) is an atavism that we can try to eliminate
because it is obviously counterproductive for our bodies.
(4) The increase in reactive oxygen species (ROS) level in mitochondria is one of
the necessary steps of the aging program. ROS cause the programmed death of
cells. This usually results in a decrease in cellularity of organs and tissues,
which leads to the weakening of numerous physiological functions and hence
to senile diseases and eventually to death.
(5) The aging program may be interrupted by removing excessive ROS from the
mitochondrial interior, a place where ROS are produced. Mitochondria can be
specifically loaded with cation derivatives of rechargeable antioxidants that
penetrate freely the mitochondrial membrane and accumulate electrophoretically inside the negatively charged mitochondria. As such antioxidants, one
can employ compounds comprising quinones and molecular electric locomotives, i.e., synthetic cations with charge being strongly delocalized over a
hydrophobic molecule (Skulachev 2003a, 2007, 2009; Longo et al. 2005;
Skulachev and Longo 2005; Severin and Skulachev 2011).
305
306
15
Until recently, the vast majority of gerontologists have considered even the first
point of the above-described concept to be completely dissident. However, the
situation has been changing rapidly over the last few years, and at the end of 2005
we managed to publish the article entitled Programmed and altruistic aging in
such a prestigious and respectable journal as Nature (Longo et al. 2005). At the
same time, we started a broad investment project on the practical use of penetrating cationic antioxidants. The main goal of the project was to try to find a way
to interrupt transmission of the aging signals from the ontogenesis-controlling
master clock to intracellular molecular targets (Skulachev 2007).
15.1
307
Fig. 15.1 Pathways of oxygen reduction in an organism. Upper scheme, four-electron reduction
of O2 to H2O, which occurs in bacterial or mitochondrial membranes. Lower scheme, oneelectron reduction of O2 results in formation of superoxide anion. This anion is protonated at
acidic pH values, while at neutral pH it is reduced to O22- in a dismutation reaction
publications in the last few years have provided data supporting the view that ROS
formation and utilization are thoroughly controlled by the organism. As a result,
ROS concentration in an organism can vary greatly depending on the functioning
of special regulating systems (Halliwell and Gutteridge 1999; Skulachev 2003a;
Tait and Green 2010; Rigoulet et al. 2011; Naik and Dixit 2011; Powers et al.
2011).
In the case of the most powerful ROS generators, such as complexes I and III
(and also complex II, dihydrolipoate dehydrogenase, and cytochrome P450), O2
formation appears to be a result of leakage of electrons that, instead of being
transferred along the respiratory chain to cytochrome oxidase (or in case of
cytochrome P450, instead of participating in substrate oxygenation), are taken by
molecular oxygen by means of its one-electron reduction. Due to the huge amount
15
Monoamine oxidase
H2O2
Oxidases of uric acid, D- and L- amino H2O2
acids
Xanthine oxidase
O2 ,
H2O2
O2
NADPH oxidase
Leakage electrons to O2
O2
O2
O2
O2
O2
Complex I
Complex III
Complex II
Dihydrolipoate dehydrogenase
Cytochrome P450
Intracellular localization
308
Concept of Aging as a Result of Slow Programmed Poisoning
15.1
309
310
15
regular oxygen pressure in air. At the same time, the rate of O2 reduction to O2 in
mitochondria depends linearly on [O2] in the entire oxygen concentration range.
That is why a 1020-fold decrease in intracellular [O2] level causes dramatic
inhibition of O2 production without a substantial inhibition of the cytochrome
oxidase reaction and hence respiration-coupled ATP synthesis. Measurements of
[O2] in animal tissues have shown it to be about one order of magnitude lower than
in air-saturated water (area B in Fig. 15.2). This area seems to be the optimal one,
as in area A [O2] is too low to support high enough rate of cytochrome oxidase
reaction, while in area C the increase in[O2] facilitates one-electron reduction of
oxygen without any benefit for the cytochrome oxidase (Skulachev 1996a).
In animal tissues (except in lungs), [O2] is maintained at a level substantially
lower than in equilibrium with air. The reason for this phenomenon is that in the
resting state the rate of O2 supply from the lungs to other organs is kept far lower
than the maximal possible. This rate increases greatly during the rest-to-work
transition. Under such a transition, lung ventilation, heartbeat, and blood flow rate
increase while blood vessels dilate. Parallel to this, the oxygen consumption by
cytochrome oxidase is increased because of the appearance of available ADP (see
Sect. 7.1). However, even under these new conditions the ratio between the rate of
oxygen consumption and supply was shown to be regulated in a way that intracellular [O2] is kept at a level no more than 10 % of its saturation in air. In
addition, the organism monitors ROS concentration in the blood by special sensors, causing constriction of blood vessels in response to increased [O2 ]
(Skulachev 1996a).
The described strategy is impossible in case of unicellular organisms. Our
calculations showed that even Azotobacter, which has the highest respiration rate
per mg protein among living organisms (see Sect. 5.2.4), cannot substantially
decrease intracellular [O2] because the distance between the bacterial cytosol and
the outer medium is so short. O2 consumption by cytochrome oxidase is immediately compensated by O2 influx from the outside. It is the polysaccharide coat
surrounding the Azotobacter cell that provides the solution to the problem in this
case. The coat prevents the convection of the liquid in the area closest to the cell
wall, and this substantially slows the movement of O2 from the medium toward the
bacterium. In the case of Azotobacter, the problem of protection against oxygen
appears to be the most crucial in media lacking ammonium salts. Under such
conditions, the very survival of Azotobacter depends on its ability to reduce
molecular nitrogen to ammonia catalyzed by a special enzymenitrogenase. This
nitrogenase is extremely sensitive to O2, and it is inactivated in the presence of
even trace amounts of O2. It is remarkable that initiation of nitrogen fixation is
accompanied by induction of a special simplified respiratory chain in Azotobacter.
This chain consumes oxygen much faster than the canonical one consisting of
complexes I, III, and IV. The simple chain (it was given the name the respiratory
protection chain) consists of only two enzymes: (1) noncoupled NADH dehydrogenase II with its only redox center (FAD) that catalyzes CoQ reduction by
NADH and (2) bd quinol oxidase that oxidizes thus formed CoQH2 by oxygen.
The second reaction generates D
lH with efficiency H+/e- = 1, i.e., twice less
15.2
311
than in the case of complex IV (see Sect. 5.2.4). As a result, the respiratory
protection chain transports only 2 H+ per NADH molecule instead of the 10 H+ of
a canonical chain. Hence, under these conditions Azotobacter has to reduce five
times more O2 to obtain the same amount of ATP (see Chap. 5, Fig. 5.6).
Escherichia coli adopted a different mechanism of protection against oxygen.
As discovered in our laboratory by Elena Budrene, these bacteria form clusters in
response to the appearance of H2O2 in the medium (Budrene and Berg 1991,
1995). Each of these clusters was shown to consist of thousands of individual cells.
Addition of hydrogen peroxide to the medium causes bacteria to start secreting
aspartic acid, which acts as the attractant. The attractant gradient serves as a signal
for bacteria to start moving toward each other. As a result, many clusters of
bacterial cells appear on a Petri dish with semisolid agar. These clusters can form
regular structures of different, sometimes very odd and regular shapes (Fig. 15.3),
which is likely to reflect uneven distribution of aspartate in the agar. One can
speculate that the outer bacterial layers in each cluster absorb oxygen so as to
decrease its concentration in the inner cluster layers. Thus, bacteria of the outer
layers are sacrificed to protect those in the interior of the cluster.
A similar strategy is used in very large muscle cells. Here, mitochondria are
accumulated right under the outer cell membrane (sarcolemma). These clusters are
312
15
15.2
313
which now can no longer reduce O2 either to H2O or to O2 . Hence, the formation
of O2 , the primary precursor of mitochondrial ROS (mROS), is inhibited.
Inhibition of aconitase also leads to intracellular accumulation of citrate, the
substrate of this enzyme. Citrate is a triply charged anion that forms complexes
with Fe2+ and Fe3+. The citrate3-Fe2+ complex undergoes irreversible autooxidation by O2, thus changing into the more stable complex citrate3-Fe3+. For this
reason, citrate accumulation under aerobic conditions decreases [Fe2+]. The Fe2+
ions are the best electron donors for reduction of peroxide ion (O22-). This process
in return entails the formation of the radical OH, the most dangerous ROS form
(see above, Fig. 15.1). Thus, inhibition of aconitase helps mitochondria (1) to
decrease O2 production and, hence, to lower the formation of all ROS that are
derivatives of superoxide, and (2) to decrease generation of OH from hydrogen
peroxide (Fig. 15.6).
314
15
15.2
315
15.2.4 Mitoptosis
Excess ROS cause mitoptosis (or suicide of mitochondria), which appears to be the
last frontier of cell protection against mitochondria that produce too much ROS
(Zorov et al. 1992; Skulachev 2003a; Skulachev and Longo 2005).
Mitoptosis triggered by ROS can develop in several different ways. One includes
pore opening in the inner mitochondrial membrane. The most likely scenario of this
phenomenon is the following. ROS oxidize the matrix-facing SH-groups of ATP/
ADP-antiporter (or some other mitochondrial anion carrier). This converts the
antiporter into a nonspecific pore (so-called permeability transition pore) that is
permeable to compounds of molecular mass\1.5 kDa. This leads to dissipation of
D
lH and all the other gradients of low molecular mass components between the
matrix and intramembrane space of mitochondria. Such a mitochondrion, similar to a
ship with all the Kingston valves left open, will inevitably perish. The mitochondrial
death will be caused not only by the efflux of NAD+, NADP+, and other water-soluble
cofactors necessary for the normal functioning of this organelle, but also by the
termination of such a vitally important reparative process as the replacement of old
damaged proteins by new active ones. There are about 600 mitochondrial precursor
proteins. One of the stages of their import from the cytosol to mitochondria involves
electrophoretic movement of their positively charged leader sequence toward the
negatively charged matrix. This process cannot occur when the pores are open and
DW = 0. Even those 1315 proteins which are encoded by mitochondrial DNA
cannot be properly arranged in the membrane because this process also requires DW
(Skulachev 1988, 1996b; Skulachev and Longo 2005).
There is one other consequence of excessive ROS production in mitochondria,
and it is also lethal for the organelle. The point is that ROS inactivate aconitase
(see above) that is normally bound to mitochondrial DNA. The inactivation
decreases the affinity of aconitase to DNA. As a result, DNA is stripped and
becomes unprotected from the damaging effects of ROS and other unfavorable
influences. Mitochondria lack histones, which protect nuclear DNA, and according
to the data obtained by R. Butow, aconitase fulfills in mitochondria a function
similar to that of histones in the nucleus (Chen et al. 2005, 2007).
It should be emphasized that the pore opening and aconitase oxidation are direct
consequences of the increase in ROS concentration in mitochondria. Both these
events require no extramitochondrial factors and are carried out by those ROS that
have been formed within the given mitochondrion. This means that this phenomenon (which we called mitoptosis) can be described in terms of suicide of the
mitochondrion that could not maintain its ROS concentration at the safe low level.
Thus, mitoptosis appears to be a mechanism for the purification of the mitochondrial
population from mitochondria that become dangerous to the cell because of their
enhanced production (or to slow removal) of ROS. Appearance of such abnormal
organelles may result, for instance, from mutations in the mitochondrial DNA.
Dead mitochondria are usually removed by autophagy. Autophagosomes play
the key role in this process. These organelles are surrounded by two membranes,
316
15
Fig. 15.6 ROS-induced inhibition of aconitase and some of its consequences. Explanations are
provided in the text
15.2
317
the organelles are partially digested, then the autophagosome fuse with a lysosome
where the digestion is completed (Narendra and Youle 2011).2
Mitoptosis is useful for a cell as long as it helps to remove a few damaged
mitochondria from a healthy group. The situation changes dramatically when the
epidemic of ROS superproduction spreads throughout the whole mitochondrial
population of the cell or the greater part of it. In this case, death of mitochondria
will inevitably lead to the death of the cell for several reasons. In the majority of
non cancer cells, glycolysis is too weak to substitute effectively for respiration as
the energy source. Moreover, mitochondria are also known to play a vitally
important role in production of certain metabolites. However, the massive dysfunction of mitochondria leads, in fact, to cell death long before the cell is depleted
of ATP and other metabolites produced by mitochondria. Here, a specific biological law seems to operate: Living organisms seek to avoid spontaneous
uncontrollable events. They do everything they can to control whatever is happening in them.
Death is the most dramatic of such events. Thus, it is no wonder that the cell
tries to control its own death, choosing to commit suicide before the damage to its
structure and functions reaches a level incompatible with life. As Monsieur Bahys,
a good-for-nothing physician in Molieres comedy LAmour medecin (Love as
a Healer) spoke, Il vaut mieux mourir selon les regles, que de rechapper contre
regles (It is better to die according to rules than to recover against the rules).
One can easily imagine the advantages that such a strategy brings to the organism.
The agony of the cell dying without any rules might damage the functioning of
the neighboring cells, and if those cells happen to release some regulatory factors,
it might influence the whole organism. The consequences can be even more tragic
if that agonizing cell manages to survive. For instance, if the agony was caused by
a ROS excess, the cell is very unlikely to have been able to protect its DNA against
ROS. As a result, the surviving cell can turn into a mutant dangerous for the
organism. Cancer cells are likely examples of this phenomenon. That is why
apoptosis as a form of programmed cell death is one of the main barriers for
malignant transformation.
The described mechanism of mitoptosis is probably not the only way for the cells to get rid of
damaged mitochondria. Boris Chernyak, Konstantin Lyamzaev, and colleagues from our group
recently discovered that a cell can gather such mitochondria in close proximity to the nucleus,
surround them with a membrane, and eject the thus formed mitochondria-containing mitoptotic
body to the extracellular space (Lyamzaev et al. 2008), see also (Lee et al. 2010; Sterky et al.
2011). This effect was demonstrated in cancer cells of HeLa line growing in the presence of an
uncoupler and an inhibitor of the respiratory chain, i.e., agents that stimulate mitochondria to
consume ATP instead of producing it. Cancer cells are known to successfully support ATP
formation not only by respiration but also by glycolysis, and using this mechanism to destroy
damaged mitochondria they can prevent the hydrolysis of the glycolytic ATP by damaged
mitochondria. It is suggested that maturation of erythrocytes as well as of crystalline lens cells,
accompanied by disappearance of mitochondria, may follow the same mechanism. This would
explain the conversion of epithelial cells into the crystalline lens cells even in the absence of
autophagy.
318
15
15.2.5 Apoptosis
Following the reasoning discussed above, programmed cell death and, in particular,
apoptosis caused by ROS, can be viewed as the next line of defense against ROS.
The term apoptosis was introduced by the Roman scholar and physician
Claudius Galen (Fig. 15.7), who noticed that the broken branch of a tree enters
winter with leaves dried up, but not fallen from it. Thus, defoliation appears to be an
active process rather than the death of leaves from cold. As Greek was the scientific
language in the Roman times, Galen defined his observation as apoptosis [from
the Ancient Greek term appsxri1 (a falling off), from the roots ap (away
from) and psxri1 (falling)]. When in 1972 Kerr and colleagues suggested that
the cell death can be programmed by its genome, they coined this phenomenon
apoptosis (Kerr et al. 1972). In 2002, the Nobel Prize in Physiology and Medicine was awarded to H. Robert Horwitz, John E. Sulston, and Sidney Brenner for
the discovery of genes that control ontogenesis. The genes of programmed cell
death were shown to be among them (for review, see Skulachev 2003b).
Apoptosis appears to be a result of a long chain of events where mitochondria
usually play the key role. ROS are one of the factors inducing apoptosis. ROS first
of all break the barrier function of the outer mitochondrial membrane3 and let
The ROS-induced opening of the already mentioned permeability transition pore in the inner
mitochondrial membrane in one of mechanisms to eliminate the outer mitochondrial membrane
as a protein-impermeable barrier. The pore opening results in disappearance of osmotic pressure
at the inner membrane since K+ and Cl-, two major ions contributing to osmotic pressure,
become permeable for the inner membrane. Now, water is distributed between mitochondrial
matrix and cytosol according to the oncotic pressure, i.e., the ratio of concentrations of high
molecular mass compounds (first of all, proteins) in the matrix and cytosol. The protein
concentration in the matrix is much higher than that in cytosol so water goes to the matrix, which
swells. Such swelling means an increase in the matrix volume due to disappearance of
15.2
319
mitochondrial proteins located in the intermembrane space go out into the cytosol.
Some of these proteins can induce apoptosis. Among them are: cytochrome
c, apoptosis-inducing factor (AIF), smac protein, protease omi, and endonuclease
G. Each of these proteins stimulates apoptosis and binds to DNA. A total of 28
positively charged amino acid residues of AIF, which are located on the surface of
the protein globule and thus can bind to the anions of DNA phosphate residues,
seem to participate in this process. As a result, DNA loses its native conformation
and becomes susceptible to nucleases. In some animals, AIF forms complexes with
endonuclease G, thus increasing its activity (Susin et al. 1999, Skulachev 2003a).
Several months later (but also in 1996) Wang and colleagues (Liu et al. 1996;
Yang et al. 1997) and independently Newmeyer in the beginning of 1997 (Kluck
et al. 1997) demonstrated release of cytochrome c to cytosol. It was found that
cytochrome c has another function besides the respiratory chain electron carrier. It
triggers a cascade of processes leading to programmed cell death. Having left
mitochondria, cytochrome c binds to a protein that was given the name apoptosis
protease-activating factor 1, or Apaf-1. This complex combines with ATP (or
better with dATP) to form a particle called the apoptosome. The apoptosome
adsorbs on its surface several molecules of the protein known as procaspase 9.
When in close proximity, procaspase 9 molecules activate each other (the procaspase 9 ? caspase 9 transition). Active caspase 9 molecules attack procaspase 3
and cleave off a part of its polypeptide chain. As a result, active caspase 3 is
formed. This protease digests a group of intracellular proteins that occupy key
positions in cell metabolism or play an essential role in the structural organization
of a cell. Internucleosomal fragmentation of nuclear DNA is one of the results of
the processes described above.
Two other proapoptotic proteins, smac and omi, which (similar to cytochrome
c, AIF, and endonuclease G) are released from damaged mitochondria to cytosol,
additionally activate the cascade triggered by cytochrome c (for reviews, see
Skulachev 2003a; Bratton and Salvesen 2010; Chipuk et al. 2010; Galluzzi et al.
2010; Orrenius et al. 2011). For instance, smac was shown to neutralize inhibitors
of apoptosis proteins (IAPs) that block caspases 3 and 9 (Du et al. 2000; Verhagen
et al. 2000).
There are at least two more mechanisms of ROS-mediated induction of apoptosis. One deals with the modification of porin, a protein of the outer mitochondrial
membrane. Normally, this protein forms pores permeable only for low molecular
mass compounds. Superoxide causes porin molecules to form oligomers that
become permeable also for much larger molecules of proteins. The proapoptotic
protein Bax seems to enhance this effect. As a result, the proteins of the mitochondrial intermembrane space enter the cytosol, and this process no longer
requires pore opening in the inner membrane.
(Footnote 3 continued)
invagination of the inner membrane (cristae). The swelling disrupts the outer mitochondrial
membrane, which has no invaginations and, hence, has a much smaller area than the inner
membrane (Skulachev 1996b).
320
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15.2.6 Necrosis
Traditionally, necrosis has been seen as opposed to apoptosis. In contrast to
apoptosis, necrosis was thought to be not programmed; it was considered to be cell
death under conditions incompatible with life. The fallacy of this statement
became obvious when necrosis (similar to apoptosis) was shown to require (i)
energy in the form of ATP and (ii) the participation of specific proteins4 (Galluzzi
et al. 2012; Vandenabeele et al. 2010; Yuan and Kroemer 2010; Christofferson and
Yuan 2010; Skulachev 2006).
Apoptosis and necrosis seem to use the same mechanisms in their early stages.
In the case of ROS, it is pore opening in the inner mitochondrial membrane. There
is also an alternative path of ROS-triggered necrosis mediated by JNK. The fundamental difference between necrosis and apoptosis is that it is only in the case of
necrosis that the outer cell membrane is destroyed and the intracellular contents
are emptied out into the extracellular space. By contrast, the outer cell membrane
retains integrity in the case of apoptosis, and the death of the cell is accompanied
by the decrease in its volume without intracellular macromolecules appearing in
the extracellular space. The cell does not advertise its suicide resulting from
apoptosis, while necrosis can be compared to a self-immolation suicide on a
crowded square. A cell in the state of apoptosis attracts attention of only macrophages that recognize it because of the appearance of phosphatidylserine in the
external layer of phospholipids of the outer cell membrane (that signals Eat
me!). In contrast to that, a necrotic cell, having burst, releases to the outer
medium many compounds that normally cannot be found there. These substances
can provoke production of cytokines by other cells. Inflammation develops as a
result. It is not a mere coincidence that one and the same stimulus can cause both
apoptosis and necrosis, the result being dependent on the quantitative parameters
When being completely depleted of ATP, the cell dies in a way different from both apoptosis
and necrosis (the so-called energetic catastrophe) (Izyumov et al. 2004; Skulachev 2006).
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of the stimulus. Thus, our experiments on the HeLa cancer cells showed that a
threefold reduction of intracellular ATP level led to apoptosis when applied for
3 h, and to necrosis when applied for a 5-h-long impact (Izyumov et al. 2004). It
looks as if a suicidal cell chooses necrosis rather than apoptosis when a negative
influence is more dangerous and lasting. An apoptotic cell might have the motto
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15.2.7 Phenoptosis
Mitoptosis, being a programmed self-destruction of malfunctioning mitochondria,
can be seen as the mechanism of mitochondrial selection, which discards potentially harmful mitochondria from the intracellular population of these organelles.
Apoptosis and necrosis fulfill the same function on the level of a cell population in
a tissue (cell selection). Then, we face the question whether this principle functions also at the level of the whole organism, i.e., whether there are some mechanisms of self-destruction of an individual which functions badly from the
perspective of a family, community, population, or evolution of the species.
The hypothetical mechanism of self-destruction of an organism we call as
phenoptosis (by analogy with apoptosis and mitoptosis (Skulachev 1997, 1999b).
Phenoptosis might protect a community of organisms from appearance of sinister
mutants or infected individuals dangerous for the community or even the whole
population (Skulachev 2003a, 2006, 2012).
In the case of phenoptosis (as well as apoptosis), the main issue is to keep the
stability (purity) of the genome (Skulachev 2003a). When dealing with complex
organisms of one and the same species that can exist for hundreds of thousands or
even millions of years, genome stability is the main guarantee of the maintenance
of the individuality of the species. Thus, it would be hardly surprising if organisms
that could not cope with protection or timely repair of their genomes were eliminated due to the activation of phenoptosis, i.e., programmed biochemical suicide
of an individual.
Examples of programmed death of bacteria and unicellular eukaryotes can be
considered as a manifestation of phenoptosis. Here, the self-destruction of a cell is
equal to the suicide of an organism, as these organisms are unicellular (Lewis
2000; Pozniakovsky et al. 2005).
The bacterial response to damage of their DNA is quite interesting. The level of
damage is monitored by a special protein that first induces the enzymes responsible
for DNA repair, and if they cannot cope with the situation, cell division is arrested. If
the damage is still there, autolysin is activated. This enzyme hydrolyzes compounds
of the cell wall, a process leading to the death of the cell. A similar mechanism has
been described in yeast cells (Lewis 2000). If DNA damage is caused by ROS, the
above-described mechanism of phenoptosis can be viewed as a way to protect the
population of unicellular organisms from poisonous oxygen species.
Sommer (1994), Manskikh (2004, 2006), and Lichtenstein (2005) hypothesized
that cancer is nothing but a phenoptosis program eliminating from the population
individuals with damaged (unstable) genome. ROS can be one of the factors
causing such instability.
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the cytokine in these experiments) (Shchepina et al. 2002). The scale of change in
ROS concentration is really remarkable. In normal cells, ROS were hardly
noticeable when a fluorescent ROS probe was used; the image obtained could be
compared to the famous Black Square by Kazimir Malevich. Induction of
phenoptosis or cell apoptosis resulted in cells glowing brightly. That is why ROS
production in such cases is often referred to as a burst. It is noteworthy that in
the presence of excess TNF, antioxidants were found to inhibit yeast phenoptosis
but showed no effect on HeLa cells apoptosis. When analyzing these results, Boris
Chernyak and colleagues from our group showed that ROS, while being unnecessary for a TNF-induced suicide of any particular cell (Shchepina et al. 2002),
serve as a lethal signal for other cells that have not received TNF. Hydrogen
peroxide was found to be the mediator of the intercellular apoptotic signal
transmission (Pletjushkina et al. 2005).
Such an effect could play an important role in tissue protection against a viral
infection, when an infected cell not only undergoes apoptosis itself, but also
stimulates suicide of the neighboring cells, the most likely potential recipients of
the virus. As a result, a dead zone is formed around the infected cell, inhibiting the
further spread of the viral infection (Skulachev 1998). Formation of such zones
was revealed in a tobacco line hypersensitive to tobacco mosaic virus. Collective
apoptosis was also shown to be a part of normal ontogenetic development of an
organism, where it is used to eliminate tissue parts and whole organs which are no
longer needed. Disappearance of the tail of the tadpole is a good example of this
phenomenon. This process is triggered by thyroid hormones. It can be studied on
tadpole tails that were cut and put into a medium of a particular composition.
Addition of thyroxin was shown to increase significantly NO production and the
level of ROS, in particular, of hydrogen peroxide. The latter causes the mass death
(apoptosis) of the cells in the tadpole tail, the size of which decreases within hours
(Kashiwagi et al. 1999). This phenomenon can be defined as organoptosis caused
by ROS (Skulachev 2002).
In certain cases, ROS act as signal molecules of less dramatic functions than a
lethal signal, first of all when mobilizing the systems of cell protection against
these ROS (an example of hormesis when pretreatment with small amount of a
poison increases resistance of an organism to subsequent treatment with much
higher doses of the same poison). For instance, the increase in superoxide production is perceived by special receptors in our organism that actuate blood vessel
constriction leading to decrease in aeration of tissues, lowering intracellular O2
concentration and, hence, decreasing the rate of superoxide production (see above,
Fig. 15.2). When speaking about the cell interior, the decrease in [O2] decreases
hydroxylation of transcription factor HIF-1. As a result, inactivation of HIF-1 is
prevented and its cleavage in proteasomes is slowed. On the other hand, the
increase in activity and quantity of HIF-1 stimulates the activity of a number of
genes encoding enzymes of antioxidant protection (Hirota and Semenza 2006). In
this way, a cell is insured against a paradoxical effect when hypoxia causes
oxidative stress. The mechanism of this stress can be explained as follows. During
hypoxia, respiration slows down because of an oxygen deficiency; this leads to
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Fig. 15.9 ROS generation during yeast phenoptosis (a) and HeLa cell apoptosis (b). a Yeast
response to amiodarone, a compound triggering the pheromone phenoptosis cascade at one of its
early stages. Colocalization of mitochondria (stained with MitoTracker Orange) and ROS
(stained with dichlorodihydrofluorescein diacetate, DCF) can be seen (Pozniakovsky et al. 2005).
b Response of HeLa cells to addition of tumor necrosis factor (TNF). Top and bottom rows show
the results of light and fluorescent microscopy, respectively (Shchepina et al. 2002)
activation of glycolysis, which in turn causes acidification of the tissue. Acidification leads to protonation of O2 , which means production of a far more
aggressive HO2. In addition, hypoxia and especially anoxia facilitate reduction of
Fe3+ to Fe2+, which actuates the Fenton reaction (H2O2 ? OH, see above
Fig. 15.1). Both these effects are not so dangerous while there is no oxygen
around. But hypoxia followed by reoxygenation (oxygen stroke) immediately
leads to formation of the most harmful compounds, HO2 and OH.
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The idea that death of an organism can be organized by this organism himself
apparently originates from Arthur Schopenhauer (Fig. 15.10) who wrote in 1818:
The individual is not only exposed to destruction in a thousand ways from the
most insignificant accidents, but is even destined for this and is led toward it by
nature herself, from the moment that individual has served the maintenance of the
species (Schopenhauer 1969, see Focher 2002 for discussion).
Alfred Russell Wallace (Fig. 15.10) was the next to indicate the possibility of
programmed death of an organism. (He is mainly known due to the fact thatat
the same time as Charles Darwin but independently from himhe postulated the
idea of natural selection.) He stated in a brief note written between 1865 and 1870
that when one or more individuals have provided a sufficient number of successors, they themselves, as consumers of nourishment in a constantly increasing
degree, are an injury to those successors. Natural selection therefore weeds them
out, and in many cases favor such races as die almost immediately after they have
left successors (quoted in Weismann 1889, p. 23). Later (in 1881), this principle
was independently formulated and developed by another great biologist, August
Weismann (Fig. 15.10): Worn-out individuals are not only valueless to the
species, but they are even harmful, for they take the place of those which are
sound I consider that death is not a primary necessity, but that it has been
secondarily acquired as an adaptation (italicized by the authors) (Weismann
1889).
Weismann was immediately accused by his contemporaries of being antiDarwinist, even though Darwin himself (Fig. 15.10) was perfectly aware of the
limitations of his hypothesis (evolution follows only those directions which are
favorable for an individual) in case of sexual selection and group adaptation. That
is what he wrote in his second famous book The Descent of Man: There can be no
doubt that a tribe including many members who were always ready to aid one
other, and to sacrifice themselves for the common good would be victorious over
most other tribes; and this would be natural selection (Darwin 1871).
Modern critics of Weismann usually quote Sir Peter Medawar, who suggested
that aging, even while being useful for a population, could not have appeared as a
mechanism invented by evolution. Medawar assumed that under natural conditions
the great majority of animals die before they become old (Medawar 1952). Today,
the falsity of this statement appears to be obvious. Aging starts long before it can
become a direct cause of the death of an individual. At the same time, it can
indirectly facilitate this death. For instance, the weakening of the organism that
progresses with age undoubtedly increases the probability of death due to attack by
predators, pathogens, etc. (Libertini 1988). Loison and colleagues (Loison et al.
1999) and independently Bonduryansky and Brassil (Bonduriansky and Brassil
2002) directly demonstrated that under natural conditions both long-lived mammals and short-lived insects suffer from aging. Such a result is not really surprising
if we take into account the fact that, for example, the reduction in skeletal muscle
mass in humans develops from 25 years of age (Lexellet et al.1988), and certain
components of the immune system start to decay even earlier (in humans, at
15 years of age, Saule et al. 2006). Decline in visual acuity begins at 30, decrease
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Fig. 15.10 Upper row Arthur Schopenhauer (left) and Alfred Russell Wallace; lower row
August Weismann (left) and Charles Darwin
in the lung volume at 35, skin elasticity around 35 (Comfort 1979; Skulachev
2009).
The hypothesis of death being the result of a program encoded by the genome
was first directly proven on the cell level (we speak here about apoptosis and
necrosis). Only very recently, programmed death phenomena have been demonstrated also on subcellular and supracellular levelsmitoptosis, collective apoptosis, organoptosis, and phenoptosis as described above.
When speaking about the different phenomena of phenoptosis, one should
distinguish between the cases of individual programmed death dependent and
independent of the life cycle of an organism. Altruistic death of a unicellular or
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from the sea to freshwater that seems to be the signal for aging in this case
(Skulachev 2009).
To what extent can the principles of accelerated aging of salmon be applied to
the slow aging of other animals? Austad believes these two phenomena are completely different in their nature and mechanism: progeria appears to be a program,
while slow aging seems to be mere accumulation of occasional damage (Austad
1997). This explanation seems to be wrong since salmon progeria is accompanied
by multiple signs of the usual aging processosteoporosis, reduction of immune
defense, appearance of tumors, thinning of the skin, reduction in skeletal muscle
mass (sarcopenia), etc. Peptides of amyloid plaques have been recently discovered
in the brain of spawning salmon, just as in the brain of old rats or humans.
The increase in the diversity of offspring seems to be one of the functions of the
above-described phenoptosis phenomena. For example, fulfillment of the phenoptosis program in the case of a female octopus or male marsupial mouse leads to
the situation when she or he can be a mother or father only once in a lifetime.
Especially impressive and well-studied cases of phenoptosis were described in
plants reproducing only once in their life. It has been known for a long time that
the death of soybean plants occurring soon after maturation of seeds can be prevented by depodding or deseeding. In both cases, the lifespan of the plant is greatly
increased. A demonstrative experiment was carried out by Nooden and Murray
(1982). When a soybean plant was depodded except for a single pod cluster in the
center of the plant, the pod cluster induced yellowing of the nearest leafs only,
whereas the rest of the plant remained green. The yellowing occurred even if the
petiole contained a zone treated with a jet of steam killing the phloem. This
treatment inactivated transport of compounds from leaf to pods occurring via
phloem (a living tissue) but not from pods to leaf, which occurs via xylem (an
already dead tissue at functional maturity of plants). This indicates that pods
induce senescence by producing a dead signal or a poison killing leaves. This
observation is in obvious contrast with a statement in a recent review by wellknown gerontologistspessimists Kirkwood and Melov (2011): There is a little
evidence that semelparous (capable of only single reproduction) organisms are
actively destroyed once reproduction is complete.
Senescence of soybeans is a fast process that takes about 3 weeks. The lifespan
of this plant is about 90 days, which is greatly increased by depodding (deseeding).
A similar increase was revealed in many other semelparous plants including Arabidopsis thaliana, the classical model species for plant physiologists and geneticists. Just this organism was recently used by Melzer and coworkers from Ghent,
who disprove one more point in Kirkwood and Melovs reasoning against programmed aging: Yet among the many gene mutations that have been discovered
that affect lifespan, often increasing it significantly, none has yet been found that
abolishes aging altogether (Kirkwood and Melov 2011). The Belgian authors
reported in Nature Genetics (Melzer et al. 2008) that a plant having mutations in the
two genes (soc1 and ful) switches from sexual to vegetative reproduction. In the
mutant, formation of seeds is inhibited. As a result, the plant does not die due to
seed-induced senescence. The lifespan of the mutants increases from 90 days to
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Fig. 15.11 Double mutant of Arabidopsis thaliana in soc1 and ful genes switches from sexual to
vegetative reproduction. In the mutant, formation of seeds (which kill the plant at age 2.5 month)
is inhibited, so the plant lives longer than 14 month, forming a woody stem. a Wild-type 2month-old plant (left) and an 8-month-old mutant (right); b 14-month-old mutant (from Melzer
et al. 2008)
Remarkably, the very fact that the death of semelparous plants is caused by their seeds literally
confirms a famous maxim of Weismann (1889) that highly organized living organisms contain
seeds of death.
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The A. thaliana case is hardly an exception. Melzer et al. (2008) mentioned that
in angiosperms, the perennial woody habit is believed to be the ancestral condition from which annual herbaceous lineages have evolved several times independently. Conversely, evolution from annual herbaceous ancestors to perennial
woody taxa has also repeatedly occurred. For example, in various annual herbaceous lineages, such as Sonchus and Echium, woody perennial species evolved on
isolated islands from their continental annual ancestors (Groover 2005; Kim et al.
1996; Bohle et al. 1996).
Among perennial plants, there are examples of organisms vegetatively reproducing for many years, then switching over to the sexual reproduction and dying
when their seeds mature.6 Several species of bamboo (Fig. 15.12) have fixed
lifespans determined by the time of inflorescence (Weismann 1889). This time is
species specific. It varies from 6 to 120 years in species that belong to a single
genus. A similar situation was described for some other perennial plants, e.g.,
Agave and the Madagascar palm Ravenala madagascariensis, flourishing in the
10th and 100th year of life, respectively, and dying after maturation of seeds
(Skulachev 2011a).
Recently, progress was made in understanding the mechanism of the senescenceinduced effect of seeds. It was shown that they somehow change the hormone
balance in leaves. Level of abscisic acid strongly increases whereas that of auxins,
gibberellins, salicylic acid, and cytokinins decreases (He et al. 2005; Shan et al.
2012; Ross et al. 2011) (see Addendum 4 for formulas of phytohormones). In the
case cytokinins, it was shown that their synthesis is inhibited (Guo and Gan 2011)
whereas their oxidative decomposition is stimulated (Price et al. 2008).
It was also found that an inhibitor of cytokinin oxidase, 6-methyl purine, retards
senescence (Price et al. 2008). For many years, it has been known that plants produce
ethylene causing defoliation (Neljubow 1901, Burg 1968). Now it is well established
that this compound activates genes involved in plant senescence (John et al. 1995;
Jing et al. 2005; Shan et al. 2012; Ross et al. 2011).
There are indications that ROS induce expression of the plant senescenceinducing genes, and antioxidants inhibit such an effect (Navabpour et al. 2003; He
et al. 2005; Barth et al. 2006). Most probably, induction of senescence, like
responses to various stress-causing factors, first initiates a burst ROS production
and then production of ethylene (Wi et al. 2010).
Thus the above-summarized observations clearly indicate at least in plants
aging can be programmed. Paradoxically, at present the majority of plant and
animal gerontologists occupy quite opposite positions concerning the aging program. The plant and animal students are sure that aging is programmed and nonprogrammed, respectively (Libertini 2012).
Such a death seems to be needed for success of sexual reproduction. Bamboo plants
reproducing vegetatively are growing so dense that seeds have little chance to find a place to give
one more young plant.
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Fig. 15.12 Bamboo phenoptosis. Botanical garden in Gteborg in the beginning of July
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population can afford to slightly change the genotype of the species by selecting
certain new properties. If these properties prove to be useful, they will eventually
be fixed in the offspring. However, if a new property has some unfavorable side
effects, it will not pass through the sieve of selection, and the experiment per se
will have no serious negative consequences for the species as the number of old
individuals in any given population is rather limited, they do not reproduce as
actively as the young ones, and they are destined to die out.
The above-described aging effect favorable for evolution suggests the brain to
age more slowly than muscles, and muscle strength weakens while reproduction is
still possible. These conditions seem to be met at least in the case of humansthe
first signs of muscle atrophy can be noted as early as at the age of 25, even though
at the beginning this process progresses slowly. Now substantial weakening of
muscle strength starts at the age of 50 for Swedish men (Larsson et al. 1979) and at
the age of 40 for men from Saudi Arabia (Al-Abdulwahab 1999). On the other
hand, at the beginning of the nineteenth century, the decrease in this parameter was
noticeable already in 25-year-old men (the studies were conducted on Belgian
men) (Larsson et al. 1979). Such dynamics can be easily explained if we take into
account the improvement of living standards that took place over the last two
centuries. In any case, the way Goldsmith puts it, due to the fact that even a
minor deterioration causes a statistically significant increase in mortality, one
should expect the evolutionary effect of aging to start in a relatively young age in
the case of wild animals (Goldsmith 2006).
Within certain limits, shortening of life can have a positive impact on evolution
rate due to the accompanying effect of the acceleration of generational change.
That is why it appears to be advantageous to regulate the rheostat determining
the rate of the aging program, so that to shorten lifespan in case of deterioration of
external conditions. And vice versa, having accomplished a major breakthrough,
i.e., having achieved a better accommodation to external conditions, a population
can allow itself to reduce the rate of evolution, i.e., to increase lifespan. That is
exactly what happened to vertebrates when they managed to conquer the airspace:
birds and bats live several times longer than terrestrial mammals of the same size,
while the intensity of metabolism in birds is 2.5-fold higher than for terrestrial
mammals of the same mass (Holmes et al. 2001). The lifespan of a bat is 17 times
longer than that of a shrew which is similar to a bat in terms of its mass and diet (it
also eats insects) (Austad 1997). In the case of birds, one can find species that live
up to 100 years of age and even longer while having no visible signs of aging
(Austad 1997).7 When turning to terrestrial vertebrates, we will see that it is those
animals that have practically no enemies that have the longest lifespan. That is the
case for giant tortoises. One of them, having been marked by Charles Darwin, was
recently still alive being fed by the zoo attendants. The problem is that the
7
An elegant experiment was done on wild albatrosses, birds living longer than 50 years.
Foraging behavior was studied using satellite tracking. It was found that old males foraged in
remote Antarctic waters, whereas young and middle-age males never foraged south of the Polar
Front (Lecomte et al. 2010).
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tortoises shell has become too heavy for this animals muscles. As a result, the
tortoise has lost its mobility and would have died in the wild because of thirst. No
signs of aging have been described also for a bowhead whale. The maximal
lifespan of this animal is over two centuries. An Aleut ruff, a northern species of a
very large marine perch, can live just as long. It is interesting to note that there are
also short-lived species among this kind of predatory fish (with maximal lifespan
of 12 years) (Austad 1997; Skulachev 2005). In any case, the fact that the lifespan
of the species of one genus can differ 100-fold indicates the presence of the
program of aging, which can hardly be explained here by mere accumulation of
random injuries. The other option seems to be more probablethe species better
adapted to the environment can afford to switch off the aging program and to
evolve more slowly.
An interesting example illustrating the presence of an aging program of major
biological significance is provided by recent studies conducted on the African fish
Nothobranchius (Terzibasi et al. 2007). The lifespan of different species of this
genus varies up to fivefold depending on their habitat in the wild. For instance, N.
furzeri, which lives in Zimbabwe in puddles formed during the rainy season and
dried after this period, was shown to have the lifespan of 34 months, which
corresponds to the length of the rainy season in this country. Nothobranchius
rachovii and N. kuhntae from Mozambique, where the rainfall is four times more,
live for about 9.5 months, while N. guentheri from Zanzibar (having a humid
climate with two rainy seasons) lives for over 16 months. In case of the shortestlived species, N. furzeri, growth and sexual maturation occur very rapidly, within
the first month of life. Then the fish mates many times and lays eggs during 2
3 months, till the puddle dries up. The next year fingerlings will hatch from the
eggs that have survived the drought. It seems to be quite remarkable that over the
short period determined by the habitat conditions (2 months), the fish manages to
age, manifesting by the end of its life a whole set of signs of senescence (slowing
of movements, loss of a research-type of behavior in an open space, osteoporosis, accumulation of lipofuscin granules in liver, a strong increase of b-galactosidase activity in skin fibroblasts, etc.). In the case of the longer living species,
completion of growth, sexual maturation, and senescence occur much later. It
seems to be significant that the described properties remain even in the case of
aquarium breeding, which means that they are determined genetically and are no
longer dependent on the current habitat conditions. It looks like the fish of the
short-lived species seek to age within that short lifespan given to them. It is also
important that the aging program has similar signs even in such distant vertebrate
classes as fish and mammals. This conservatism presents clear evidence of operation of a program.
It was Nature Herself that conducted an interesting experiment in the headwater
of the rivers Oropouche and Yarra (Trinidad). Reznik and colleagues (Reznick
et al. 2004) discovered the headwater of these rivers to be cut from their downstream by waterfalls impassable for predatory fish (in particular, for pikes) feeding
on guppies. As a result, guppies living in the headwater had practically no natural
enemies, while those from the downstream were constantly facing the danger of
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being eaten by pikes. The researchers caught guppies from the different areas and
started to breed them in aquariums with no enemies present. The fish of the second
aquarium generation (from the group that used to coexist with pikes) were shown:
(1) to live 35 % longer, solely due to the increase in the reproductive life period;
(2) to reproduce twice as fast, and (3) to respond to danger 1.5 times faster (similar
to that clever hare in the example discussed above). An increase in the first two
parameters is favorable for higher fertility of the brood under conditions of constant loss of fish due to the attacks of the predator, while the third decreases such
losses. Overall, these data contradict one of the key postulates of classical gerontology that fertility can be increased only if the lifespan is reduced.8
When analyzing the above relationships, one can conclude fertility and lifespan
to be regulated by, so to say, two rheostats, which sometimes react differently to
the changes of the habitat conditions. A population uses these types of regulations
not only for survival, but also for changes in the rate of evolutionunder unfavorable conditions evolution is accelerated due to the increase in fertility and
hence, the diversity of offspring. Parallel to that, lifespan can be shortened under
the influence of habitat pressure as well as internal factors of an organism. It is
important to stress that this phenomenon cannot be explained by an assumption
that the organism spends too many of its resources for reproduction and, hence,
becomes short-lived (as suggested by Kirkwood and Austad 2000). Within certain
limits, the reduction of lifespan can favorably affect the pace of evolution due to
acceleration of alternation of generations. That is why it appears to be advantageous to regulate the rheostat that determines the pace of the aging program for
a shorter life under worsening external conditions. Having attained new frontiers,
i.e., having reached better accommodation to external conditions, a population can
reduce its evolutionary pace. The above-described case of two populations of
guppies seems to be quite peculiar in this respect. When facing the threat of
immediate extinction, the population had to use all the available resources by
increasing both fertility and duration of reproductive lifespan (Skulachev 2005).
Reznik was so sure about the lifespan in hell being much shorter than in heaven, that,
when starting his long-term project, he published his prediction of this result (Reznick 1997). In
their recent paper, Reznik and colleagues no longer quote their erroneous predictions (Reznick
et al. 2004).
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Compare with the data of Kirkwood and coworkers on the positive correlation between the
lifespan of different mammals and the resistance of their fibroblasts to oxidative stress (Kapahi
et al. 1999).
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skin, skeletal muscles, and kidneys, but not in brain and heart. This corresponds to
the content of p66shc in different organs, which is the lowest in brain and heart,
i.e., in those organs that are the last to age.
In 1971, the Russian gerontologist Vladimir Dilman (Fig. 15.14; Dilman 1971;
1978; Dilman and Anisimov 1979) postulated that aging is controlled hormonally,
insulin being the factor that stimulates aging. Later, this hypothesis was confirmed
by research carried out on eukaryotes of different taxonomic groups. Blcher and
colleagues (Bluher et al. 2003) showed the knockout of insulin receptor in adipocytes prolonged the lifespan of mice by 18 %. Independently, Holzenberger and
colleagues (Holzenberger et al. 2003) showed that mice heterozygous for the
receptor of insulin-like growth factor I lived 26 % longer than the wild type. The
effect was accompanied by a decrease in [p66Shc] by 50 %. The Ras protein and
Akt/PKB-protein kinase phosphorylating the serine and threonine residues of
proteins were shown to be part of the system responsible for the shortening of the
lifespan of the mice (Longo and Finch 2003).
Some data indicating insulin-like factors shorten lifespan were also obtained
with flies and nematodes. In the case of C. elegans, a mutation of the daf-2 gene
prolonged life 23-fold (Lakowski and Hekimi 1996). NAD+-dependent histone
deacetylase (SIR2.1 protein) was shown to suppress one of the regulatory cascades
of insulin and to prolong the lifespan of these animals. In case of yeast, the
increase in sir2 gene dose prolongs their life. Longo and Finch showed that the
deletion of gene Ras2 and an Akt gene analog cause 23-fold increase in the yeast
lifespan (Longo and Finch 2003).
There is much evidence for the participation of mitochondrial DNA damage in
the regulatory cascade that causes aging of both yeast and animals. This statement
has been recently directly proved in elegant experiments carried out in the
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soldiers, a queen, and her several breeding partners. Only the queen and her
husbands breed. Under laboratory conditions, the naked mole-rats die at the age
of 2528 years for unknown reasons (Buffenstein 2005). The level of their mortality does not depend on age. An aging program seems to be not operative in these
mammals. The program is most likely to be interrupted downstream of ROS
formation, because the level of ROS production during reverse electron transfer
and the degree of oxidation of biopolymers in naked mole-rats are higher than in
mice (Labinskyy et al. 2006; Buffenstein 2005; Lambert et al. 2007). These
observations are not really surprising if we take into account the fact that superoxide dismutase and catalase activities are similar in these two species, while the
activity of the third most important antioxidant enzyme, glutathione peroxidase, is
70 times lower in a naked mole-rat. The fact that the cells of the naked mole-rat are
extremely resistant to H2O2-induced apoptosis can provide an explanation of this
paradoxical situation (Labinskyy et al. 2006). In this respect, the naked mole-rat is
like the long-lived mice carrying the mutations in the genes encoding the p66shc
protein: their cells are also resistant to apoptosis induced by ROS (in this chapter,
see Sect. 15.4.4 above).
The absence of some pain sensors is another specific feature of naked mole-rat.
Park and colleagues (Park et al. 2008) recently showed that these rodents lack a
small peptide of 11 amino acid residues called the P substance; in other animals, it
participates in the transmission of the pain signal induced by capsaicin, a potent
inhibitor of complex I of the respiratory chain (see Sect. 4.2.4 and Appendix 3). It
seems possible that insensitivity of naked mole-rats to capsaicin is related to their
ROS resistance since capsaicin, like other complex I inhibitors, should increase
ROS production by mitochondria oxidizing NAD-linked substrates (Skulachev
1996a). Sensitivity to capsaicin can be restored by injecting DNA encoding the P
substance into a naked mole-rats leg. This treatment does not influence the other
legs, which were still insensitive to the pain inducer. Apparently, the naked molerat queen and her husbands do not interact with external pain sources. If this
is the case, then the mutation interfering with the process of signal transfer from
skin pain receptors will be neutral and will avoid being eliminated by natural
selection. The aging program, which according to our hypothesis provides evolutionary advantages under the pressure of natural selection only in the presence of
enemies, might be switched off in a similar manner. We need to remember that the
queen, the only individual bearing offspring, and hence, participating in the evolutionary process, has practically no enemies. She is protected from them by living
in the vast underground labyrinths guarded by the army of numerous soldiers
who, when in the wild, live not longer than 3 years and die because of fighting with
other colonies of naked mole-rats or with snakes that creep into their holes. Quite
recently, Gladyshev and his colleagues (Kim et al. 2011) published the complete
sequence of the naked mole-rat genome and some data on age-related changes in
its expression. In particular, expression of 54 genes of brain was investigated. In
humans, 33 genes among these 54 genes were underexpressed and 21 overexpressed during aging. Of these, 32 genes did not show consistent expression
changes with aging in naked mole-rat, including 30 genes that had stable
342
15
expression and two genes that changed in the opposite direction to human brain (4year-old and 20-year-old mole-rats were compared). These results clearly show
that expression of a large group of genes in the naked mole-rat brain is, in fact,
age-independent in contrast to that in human brain.
15.4
343
decrease that seems to be the earliest sign of aging, as in the case of humans it can
be observed starting from 7 years of age (Austad 1997).
On the whole, the hypothetical aging program of mammals can be presented as
follows (Fig. 15.15). It is assumed that aging represents the final step of ontogenesis, resulting from a slow controlled poisoning of the organism by endogenous
toxins, namely ROS. This intoxication increases with age and is initiated by a
signal generated by the master clock, being mediated by a decrease in the level of
juvenile hormone(s) and by an increase in level of age-inducing hormone(s) (step
1). Such a change in hormonal balance somehow increases ROS level specifically
in mitochondria (step 2), which in turn entails stimulation of apoptosis whose rate
becomes higher than the rate of proliferation if we deal with proliferating tissues
(step 3). This situation leads to a decrease in number of cells in the tissue
(cellularity) (step 4), inevitably resulting in a decay of functions of organs (step 5)
(Skulachev 2003a, 2009).
344
15
this case aging seems to be stimulated at stage 3 (see Fig. 15.15) (Skulachev
2003a).
15.4
345
vitamin E. That is why synthetic, rather than natural, antioxidants that seem to be
better candidates for a tool to cancel the aging program. These antioxidants need to
be specifically addressed to mitochondria, as their primary targets should be
intramitochondrial ROS. Thus, the question is how to provide addressed targeted
delivery of antioxidants to mitochondria.
In the late 1960s, hydrophobic cations and anions with their charge strongly
delocalized over a relatively large hydrophobic molecule were described in our
group in cooperation with the group of Efim Liberman (Fig. 15.16) (Liberman
et al. 1969; Liberman and Skulachev 1970). Later these compounds were named
Skulachev ions (Sk+ and Sk-) by the well-known American biochemist David E.
Green (Green 1974). Due to low level of their hydration (water dipoles cannot
form a water coat around an ion if its charge is delocalized), Sk+ and Sk- can
easily penetrate membranes. As a result, their distribution between two compartments separated by a membrane follows the Nernst rule (10-fold gradient of ion
concentration per 60 mV of DW) (Liberman and Skulachev 1970). As any ion
gradient depends logarithmically on DW, a mitochondrion with DW = 180 mV
(negative inside) is capable of maintaining a 1000-fold gradient of Sk+ or Sk-. The
tetraphenylphosphonium cation and tetraphenylboron anion were studied as
examples of Sk+ and Sk-, respectively. Later, Sergey Severin, Lev Yaguzhinsky,
and one of this books authors (V.P.S) (Severin et al. 1970) suggested that ions of
Sk+-type can serve as molecular electric locomotives: if a compound X needs to
be delivered to mitochondria, it would be enough to have it bound to Sk+, for
mitochondrial matrix is the only intracellular compartment that is charged negatively in comparison with cytosol (Liberman and Skulachev 1970).
Such logic was applied when we tried to explain the role of the carnitine cation
group in the transport of fatty acid residues into mitochondria (Severin et al. 1970;
Skulachev 1988). It was also used by Murphy and Smith for the construction of
mitochondria-targeted antioxidants (Burns et al. 1995). Later, Murphy mainly used
for his experiments the compound called MitoQ with ubiquinone serving as the
346
15
antioxidant, and decyl triphenylphosphonium cation as the Sk+ (Smith et al. 1999;
Kelso et al. 2001, 2002; James et al. 2005; Saretzki et al. 2003; Jauslin et al. 2003;
Murphy and Smith 2007).
We confirmed Murphys data that micromolar concentrations of MitoQ are able
to accumulate in mitochondria and to protect them from oxidative stress. But at the
same time, Mikhail Vysokikh in our group (Skulachev 2007; Antonenko et al.
2008; Skulachev et al. 2009) showed that even a small overdose of this compound
causes the opposite effect: MitoQ was shown to turn into a powerful prooxidant
catalyzing H2O2 generation in mitochondria, the rate of this process being so high
that it becomes comparable with the respiration rate of mitochondria in the State 4.
Similar observations were reported in three other laboratories including Murphys
group (James et al. 2005; OMalley et al. 2006; Doughan and Dikalov 2007). This
is why we attempted to find an antioxidant much better than ubiquinone and
combine it with Sk+. These experiments proved to be successful (see Chap. 16).
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Chapter 16
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356
16.1
357
decyltributylammonium (in SkQ4), and also similar derivatives of methylplastoquinone (in SkQ3), of ubiquinone (in MitoQ), desmethoxyubiquinone (DMQ), and a
number of others have been used as Sk+ in these compounds. (Antonenko et al. 2008a;
Skulachev et al. 2011). The penetrating ability of these compounds was studied by
Inna Severina, Yury Antonenko, and coworkers in artificial bilayer phospholipid
membrane (BLM). This ability was shown to decrease is the series: SkQR1 [ SkQ1,
SkQ3, MitoQ [ SkQ5 SkQ4 (Antonenko et al. 2008a; Rokitskaya et al. 2008).
Antioxidant properties tested in aqueous solutions, BLMs, liposomes, linoleic acid
micelles, and mitochondria followed the pattern SkQR1, SkQ1 [ SkQ3,
DMQ [ MitoQ (Antonenko et al. 2008a, b). Antonenko et al. found that the prooxidant properties of these followed the opposite pattern, reaching their maximal value in
MitoQ (Antonenko et al. 2008a). On the basis of these data, SkQ1 and its fluorescent
derivative SkQR1 were chosen for further studies
In in vitro experiments on heart mitochondria, Vyssokikh et al. showed that the
respiratory chain was able to reduce SkQ1. This means that this compound can act
as a rechargeable antioxidant. The bH heme of complex III was found to be the
site of reduction of SkQ1. This heme is located close to the inner surface of the
inner mitochondrial membrane. The reduction of SkQ1 was completely inhibited
by antimycin (Antonenko et al. 2008a; Skulachev et al. 2009).
358
When using SkQR1, Chernyak and his colleagues showed the SkQ-type compounds added to the cell culture are selectively accumulated in mitochondria
(Figs. 16.3, 16.4a) (Antonenko et al. 2008a; Skulachev et al. 2009). Then, the
studied compounds were shown to inhibit the H2O2-induced apoptosis of HeLa
cells and human fibroblasts (activity: SkQR1 [ SkQ1 [ MitoQ; Fig. 16.4c)
(Antonenko et al. 2008a; Skulachev et al. 2009). The uncoupler FCCP abolishing
Dw and preventing SkQ1 accumulation by mitochondria (Fig. 16.4a) inhibited the
antiapoptotic effect of SkQ1 (Fig. 16.4b). Induction of apoptosis by H2O2 was
found to have two consequences soon after its addition: strong increase in production of ROS by cells (see DCF probe for ROS, Fig. 16.4d) and decomposition
of mitochondrial filaments to small roundish mitochondria (Fig. 16.4e). Both these
effects were prevented by SkQ1. C12TPP, an SkQ1 analog without plastoquinone,
was ineffective (Fig. 14e). Chernyak et al. showed that ROS-induced necrosis was
also sensitive to SkQ1 (Antonenko et al. 2008a; Skulachev et al. 2009). Decylplastoquinone (DPQ) and tetraphenylphosphonium failed to substitute for SkQ1,
whereas antioxidants N-acetylcysteine (NAC) and Trolox were effective but at
very much higher concentrations (Fig. 16.4f). SkQ1 was shown to prolong the
median lifespan of the mycelial fungus Podospora anserina, the crustacean Ceriodaphnia affinis, the insect Drosophila melanogaster, the fish Nothobranchius
furzeri, and mice (in case of mice the median lifespan was doubled; Fig. 16.5).
Vyssokikh, Filenko, Pasyukova, Shidlovsky, Anisimov et al. took part in these
studies (Skulachev et al. 2009; Anisimov et al. 2008, 2011). In all the studied
cases, SkQ1 was the most effective in reduction of mortality at early stages of
aging (the first 20 % of animals to die), but it showed little effect on the maximal
lifespan and the last 10 % of deaths. Anisimov et al. showed that in the case of
mice in a nonsterile vivarium, SkQ1 added to drinking water (0.55 nmol/kg per
day) strongly lowered mortality caused by infectious diseases but showed little
effect on mortality caused by cancer (Anisimov et al. 2008; 2011; Skulachev et al.
2009). In studies on mice and rats, the groups of Kolosova, Anisimov, Drize,
Cannon, and some others showed that SkQ1 decelerates the development of such
16.1
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360
b Fig. 16.4 Effects of SkQ1, SkQR1, and MitoQ on human cell cultures. a Kinetics of SkQR1
accumulation in HeLa cells and its efflux from cells. Cells were treated with 50 nM SkQR1 at
zero time. Uncoupler (10 lM FCCP) was added where indicated. SkQR1 accumulation and efflux
were measured fluorimetrically in a flow cytometer. b Incubation of human fibroblasts for 7 days
with 20 nM SkQ1 prevents H2O2-induced apoptosis. Apoptotic cells were counted 24 h after
addition of H2O2. Additions: 1 lM FCCP, 400 lM H2O2. c SkQ1 and SkQR1 are more efficient
than MitoQ in arresting H2O2-induced apoptosis in human fibroblasts; for the experimental
conditions, see b. d SkQ1 (20 nM, 7 days) prevents accumulation of H2O2 by H2O2-treated HeLa
cells (200 lM, 45 min treatment). The cells were stained with 4 lM DCF for 15 min before
being analyzed in a flow cytometer. e Preventive effects of SkQR1, SkQ1, and MitoQ and
ineffectiveness of C12TPP (an SkQ1 analog without the quinone moiety) on H2O2-induced fission
of elongated mitochondria. Human fibroblasts were preincubated with SkQR1, SkQ1, MitoQ, or
C12TPP for 2 h and then treated with 400 lM H2O2 for 3 h. f SkQ1 prevents necrosis caused by
illumination of HeLa cells stained with Mitotracker Red. The cells were illuminated for 15 min
with the green light (545 nm) so as to deliver the light energy of 34.8 J/cm2. Necrosis was
measured 5 h after the illumination. The cells were treated with 1 lM SkQ1, the mixture of 1 lM
tetraphenylphosphonium (TPP) and 1 lM decylplastoquinone (DPQ), 20 mM N-acetylcysteine
(NAC), or 1 mM Trolox 1 h before illumination (Skulachev et al. 2009)
Age-related involution of the thymus is the most demonstrative example of aging being
programmed. This effect surely cannot be explained by a mere accumulation of injuries in an
aging organism. Involution of the thymus, the organ producing T-lymphocytes, one of the types
of key cells of the immune system, undoubtedly leads to weakening of immunity. These changes
are aggravated by the parallel involution of the spleen follicles, where the other very important
type of immune cells is produced, B-lymphocytes. SkQ decelerates (but does not cancel) both of
these processes (Obukhova et al. 2009).
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oxidative stress) was observed with SkQ1 but not with the same dose of MitoQ or
even with much larger dose of a-tocopherol (Fig. 16.7a, b).
In fibroblasts obtained from control and life-long SkQ-fed mice, SkQ1 was
shown to prevent the age-related appearance of b-glucosidase activity and phosphorylation of H2AX histone (Anisimov et al. 2008; Skulachev et al. 2009).
Studies carried out on the same model by Spivak and Mikhelson demonstrated the
age-related increase in spontaneous apoptosis and its stimulation by the addition of
hydrogen peroxide. Both effects were canceled by SkQ1 treatment (Anisimov et al.
2008; Skulachev et al. 2009). It is important that SkQ1 did not arrest apoptosis
completely, but prevented its threefold increase in old mice. Exactly, this type of
effect might be expected within the framework of our scheme (see Fig. 15.15). A
demonstrative effect of in vivo treatment with SkQ1 was observed in Kolosovas
group in Novosibirsk, where age-dependent behavioral changes were studied in
Wistar rats (Stefanova et al. 2010). Rats of 3- and 14-month age were investigated.
An elevated plus maze with two open and two closed arms was used. Young
362
Fig. 16.7 SkQ1 prevents development of cataract and retinopathy in OXYS rats. SkQ1 and other
compounds were administered per os. a, b 3-month-old rats; -tocopherol (670 lmol/kg body
mass/day), MitoQ (250 lmol/kg/day), or SkQ1 (250 lmol/kg/day) was administrated during the
last 1.5 months (Zinovkin et al. 2013). Electroretinograms of three OXYS rats: 1 3 months, no
SkQ1 (red); 2 24 months, no SkQ1 (black), and 3 24 months, 250 nmol SkQ1/kg per day with
food (green). (Skulachev et al. 2009)
animals placed in the maze center entered both types of arms with equal probability. However, the 14-month-old rats preferred to enter the closed arms only, the
probability of entering the open arms being negligible. Addition of a very small
amount of SkQ1 to the food (250 nmol/kg body mass daily) for the last 10 weeks
completely reversed the age effect. With SkQ1, the probability of entering an open
arm for the 14-month-old rats was as high as for young rats and, when entering an
open arm, the SkQ1-treated old rats spent in it a time which was almost as long as
for the young rodents (Fig. 16.8). The number of squares crossed by the animals in
the open field test was slightly (by 25 %) smaller in the 14-month-old rats than for
the 3-month-old animals. This difference was also abolished by SkQ1. Rearings in
the maze or in the open field and head dips in the maze were age independent and
SkQ1 insensitive.
In a number of cases, SkQ was shown to retard the development of accelerated
agingprogeria. Such results were obtained on OXYS rats. SkQ1 addition to food
(250 nmol/kg) was shown not only to prevent (Fig. 16.7) the development of early
cataract and retinopathy, but also prevented peroxidation of cardiolipin, accumulation of malondialdehyde and carbonylated proteins in tissues (Kolosova,
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Zinovkin et al.) (Antonenko et al. 2008a; Skulachev et al. 2009; 2010; Skulachev
et al. 2011). In case of cataract and retinopathy, eye drops containing
2.5 9 10-7 M SkQ1 cured already developed disease within 1.5 months
(Antonenko et al. 2008a; Skulachev et al. 2009). This therapy was also effective in
the case of cataract in normal Wistar rats. However, Wistar rats hardly ever live
long enough to experience retinopathy, so in this case we had to change the object
of research. The experiment carried out by Kopenkin, Sotnikova, and their
coworkers (Antonenko et al. 2008a; Skulachev et al. 2009, 2011) from the
Skryabin Veterinary Academy, Moscow showed the SkQ1 eye drops to be
effective in treating retinopathy in dogs, cats, and horses. A total of 271 animals,
for which all the traditional methods failed to have positive effect, received SkQ1.
The most striking positive effects were achieved in cases of congenital dysplasia of
the retina (dramatic improvement in 67 % of cases) and its secondary degeneration
(54 %). In the case of progressive retinal degeneration, SkQ1 was less effective
364
(29 %). In 89 cases, animals that had recently become blind were the object of
research. Of these, 67 animals regained their sight after being treated with SkQ1
eye drops for 26 weeks.
These positive results are in contrast to the failure of Murphys group, where
congenital retinopathy in mice was treated with MitoQ (Vlachantoni et al. 2006).
The likely reason for this failure is the effective MitoQ concentrations (i.e. those
having antioxidant effect) being too close to those causing a prooxidant effect. The
authors, however, assumed that the studied types of retinopathy might have been
resistant to antioxidants, which is also possible.
When completing this series of experiments, we studied artificially imposed eye
diseases, namely, experimental uveitis (Senin et al., see Neroev et al. 2008;
Skulachev et al. 2009) and glaucoma in rabbits (Erichev and Iomdina, see Neroev
et al. 2008; Skulachev et al. 2009). Both of these diseases are known to be closely
connected to very strong oxidative stress, and in both cases SkQ1 eye drops were
shown to have therapeutic effect. Experiments on treatment of uveitis were
especially demonstrative. Immunization of rabbits with arrestin, a protein specific
for photoreceptor cells, caused the development of this autoimmune disease, which
eventually led to blindness. Then, one eye was treated four times daily with drops
of 2.5 9 10-7 M SkQ1. After several days, the animals started to regain their
sight, but only in the eye that was treated with SkQ1. The same procedure prevented the development of uveitis if the eye drops were used during the immunization. The treatment was successful in 100 % of cases, both in prevention of
uveitis and in treatment of the already developed disease.
Obukhova et al. studied progeria in OXYS rats, where SkQ1 prevented the agedependent acceleration of decrease in number of cells in thymus and spleen primary follicles that are responsible for the formation of T- and B-lymphocytes,
respectively (Obukhova et al. 2009). Ryazanov in the USA studied radiationinduced progeria, and SkQ1 canceled such aging sign as graying of hair (black
mice were used for these experiments).
Results obtained on mutant mice with alanine substituted for aspartate-257 in
the proofreading domain of mitochondrial DNA-polymerase c are interesting.
(These mutants have been already discussed above in connection to the role of
mitochondria in aging, see Chap. 15). Addition of SkQ1 to drinking water
increased not only in lifespan but also healthspan of such animals. SkQ-treated
mutator mice died without the whole set of senile traits that could be observed
in mutator mice that did not receive SkQ1. Among these traits were osteoporosis (lordokyphosis), loss of hair on the cheeks and body, depression, decrease in
body temperature, disappearance of estrous cycles, etc. The effect of SkQ on
mortality was developed in spite of the fact that mice were kept under sterile
conditions (Cannon and coworkers, Sweden).
Experiments performed in the groups of Kapelko and Pisarenko (Bakeeva et al.
2008; Skulachev et al. 2009) and the group of Zorov (Bakeeva et al. 2008;
Skulachev et al. 2009; Plotnikov et al. 2010) showed that SkQ1 or SkQR1 treatment reduced considerably an unfavorable effect of ischemia and subsequent
reperfusion on heart rhythm in rats; SkQ1 also reduced the areas of myocardial
16.1
365
infarction and brain stroke. Both of these compounds prevented the death of rats
who had one kidney removed and the other one having undergone 90-min-long
ischemia (Skulachev et al. 2009; Plotnikov et al. 2011) (Fig. 16.9). SkQR1 was
also effective in the case of pyelonephritis and rhabdomyolysis (Plotnikov et al.
2010, 2011). In experiments on p53-/- mice performed in the group of Boris
Kopnin, SkQ1 as well as nontargeted antioxidant N-acetylcysteine (NAC) slowed
development of lymphoma, reduced ROS level in spleen, and prolonged lifespan,
the effective SkQ1 concentration being as low as 5 nmol/kg per day. To reach a
similar effect, 6 mmol NAC/kg per day was needed, i.e., 1.2 million times more
than SkQ1 (Agapova et al. 2008; Skulachev et al. 2009). SkQ1 was even more
effective in the case of rat cardiac arrhythmia and in prolongation of the lifespan of
mice: SkQ1 concentrations of 0.05 and 0.5 nmol/kg per day caused the maximal
effect (Bakeeva et al. 2008; Skulachev et al. 2009).
There are several reasons for such high efficiency of SkQ. SkQ1 can accumulate
electrophoretically in the cell cytosol reaching a level about ten times higher than
outside the cell. It is the electric potential difference on the outer cell membrane
that causes this accumulation (DW of about 60 mV). Even much more significant
(1000-fold) accumulation takes place in mitochondria (DW of about 180 mV). The
distribution coefficient in a membrane/water system is about 10,000 for SkQ1.
Because of this, SkQ1 concentration in the inner half-membrane layer of the inner
mitochondrial membrane should be 10 9 103 9 104 = 108 times higher than in
the extracellular space (Skulachev et al. 2009; 2010).
The mechanism of the antioxidant effect of SkQ can involve two types of
phenomena, i.e., (1) quenching of radical intermediates of lipid peroxidation and
(2) mild uncoupling. SkQH2, when accumulated in the inner half-membrane layer
of inner mitochondrial membrane, can quench peroxyl radicals of polyunsaturated
fatty acids (LO2) of phospholipids forming this layer (especially of cardiolipin)
(Fig. 16.10). Most probably, LO2 is reduced by SkQH2 to fatty acid peroxide
(LOOH), and a chain reaction of oxidative damage to mitochondrial lipids is
interrupted:
LO2 SkQH2 ! LOOH SkQ H :
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Fig. 16.10 Conversion of linoleate residue (LH) into its primary radical form (L), peroxyl
radicals (LOO), and peroxides (LOOH). R, a radical initiating the chain reaction of lipid
peroxidation (OH, HO2, L, etc.)
3
SkQ cytochrome b2
H 2 H ! SkQH2 cytochrome bH :
16:2
16.1
367
2011). Our data can be compared to the recent results obtained by Padalko and
Kowaltowski (Padalko 2005; Caldeira da Silva et al. 2008) using low dinitrophenol concentrations, which increased the lifespan of Drosophila and mice as
well as prevented oxidation of DNA and proteins during aging. Mild uncoupling
may be the second antioxidant mechanism of SkQs. It requires somewhat higher
SkQ1 concentrations and differs from direct antioxidant effect of SkQ1 described
by Eq. (16.1) in that C12TPP is also active.
Table 16.1 summarizes our results obtained in studies of effects of SkQ1 and
SkQR1 on animals in vivo. SkQ-type compounds were shown to decelerate
26 different traits of senescence, this showing that our compounds are powerful
geroprotectors. It should be emphasized that all effects of SkQs occur at their
nanomolar and in some cases even subnanomolar concentrations per kg body mass
daily, which is much lower than for other known geroprotectors. It is noteworthy
that they not only decrease mortality at the early and middle stages of aging, but
also prevent the development of a large number of senile defects. In other words,
SkQs can prolong healthspan.2 It is clinical trials that will determine to what an
extent a similar effect will be observed in humans, and these trials have already
started.
Clinical trials of SkQ1 are already completed for dry-eye syndrome. 3-week
administration of SkQ1-based eye drops were shown to result in significant
increase in (1) amount of tears produced, (2) tear meniscus height, (3) visual
acuity, etc. In the double-blind trial, SkQ1-based eye drop efficacy was compared
to that of Tears Naturale (Alcon), a standard medicine recommended for light and
moderate dry-eye syndrome treatment. The SkQ1-based drops proved to be much
better than Tears Naturale. The dry-eye traits completely disappeared in 60 and
20 % patients after 3 weeks treatment with SkQ1 and Tears Naturale, respectively
(Fig. 16.11) (Yani et al. 2012). In December, 2011, the Ministry of Health of
Russia issued permission to produce and sell in drugstores Visomitin eye drops
containing 250 nM SkQ1. The new drug is available in Russian drugstores since
2012.
2
When speaking about prolongation of youth, we would like to stress that canceling of an aging
program does not mean personal immortality of an individual. If we look at the aging program as
a biological mechanism accelerating evolution, then increase in the maximal lifespan is rather
unlikely to be an indispensable consequence of canceling of such a program. The input of the
most long-lived individuals into the general productivity of the population is rather small because
of their paucity. Moreover, age-dependent genome damage may accumulate not only because of
the programmed increase in ROS, but also for other reasons. This process is particularly
dangerous for females: DNA of their oolytes has to remain intact through the whole life of an
individual. Thus, it is not surprising that at a certain age females lose their ability to reproduce.
Menopause begins, which is particularly long in the case of humans and whales, but it is present
even in Drosophila. It is no coincidence that in the early and middle stages of aging the
programmed death input in mortality rate is rather large, and its SkQ-induced canceling causes
substantial increase in lifespan. Further aging brings forward other causes of death that do not
depend on the aging program and hence cannot be canceled by SkQs. Cancer is one of the main
causes of death of this type. It seems to be also a phenoptotic program, but of a different function,
i.e. elimination of individuals with overloaded damage to the genome.
Mutator mice
Mole-vole
Dwarf hamster
Mice
Rats of OXYS-line
(continued)
Table 16 1 SkQ decelerates programs of aging (slow phenoptosis) and death after crisis (acute phenoptosis) in animals
Result
Species
Studied treatment
References
368
16 Possible Medical Applications of Membrane Bioenergetics
Mutator mice
Mutator mice
Mice
Rats
Mice
Rats of OXYS-line
Rats of OXYS-line
Mice
Mice
Mice
Mice
Mice
Mice
Species
Table 16 1 (continued)
Result
of SkQ1
of SkQ1
of SkQ1
of SkQ1
of SkQ1
of SkQ1
of SkQ1
of SkQ1
of SkQ1
of SkQ1
of SkQ1
Life-long administration
with drinking water
Life-long administration
with drinking water
Life-long administration
with drinking water
Life-long administration
with drinking water
Life-long administration
with drinking water
Life-long administration
with drinking water
Life-long administration
with drinking water
Life-long administration
with drinking water
Life-long administration
with food
Life-long administration
with food
Life-long administration
with drinking water
Studied treatment
References
16.1
369
Black mice
Rats with one kidney
19-months-old rats
Rats of OXYS-line
Rats of OXYS-line
Mice
References
(Stefanova et al. 2010)
(continued)
Studied treatment
Administration of SkQ1 with
drinking water for last
10 weeks
Life-long administration of SkQ1
with drinking water
Life-long administration of SkQ1
with food
Species
Rats
Table 16 1 (continued)
Result
370
16 Possible Medical Applications of Membrane Bioenergetics
References
Rats
Studied treatment
Single injection of SkQR1 before
the brain ischemia
Species
Rats
Table 16 1 (continued)
Result
16.1
SkQ Decelerates the Aging Program
371
372
16.2
373
experiments on albino mice that a 2-day long fasting every week is sufficient to
extend their lifespan by 5060 %. Carr et al. (1949) observed, again on mice, that
the reproductive ability (which was lost by the end of the first year in the case of
unrestricted feeding) was preserved until at least the 21 months of life when the
mice were limited in food over the first 1115 months, and then received food ad
libitum. According to the data of Stuchlikova et al. (1975), rats, mice, and golden
hamsters experiencing food restriction by 50 % over 2 years lived 20 % longer
than the control. Dietary restriction during the first year of life only prolonged the
lifespan by 4060 %, and during the second year by 3040 %.
Further research showed that the effect of dietary restriction involves both the
carbohydrate and protein components of food. The effect of proteins is associated
with only one amino acidmethionine (Richie et al. 1994; Miller et al. 2005; Sanz
et al. 2006; Caro et al. 2009). Methionine belongs to the group of essential amino
acids, so food is the only source of this compound. It was found that a diet in
which proteins were replaced by a mixture of amino acids containing no methionine not only favors a longer life, but also decreases mitochondrial ROS generation and oxidative damage to mitochondrial DNA (Sanz et al. 2006; Caro et al.
2009). Interestingly, dietary restriction has no effect on the oxidation of nuclear
DNA (Edman et al. 2009), which might be expected by the generally accepted
view on aging as a nonspecific age-related ROS damage to DNA and other cellular
biopolymers.
In our opinion, calorie restriction is perceived by an organism as a worrying
signal of food shortage (Skulachev 2011). Even partial starvation may entail
decreased fecundity. And this, in turn, jeopardizes the very existence of the
population. To prevent this situation, at least partially, it is sufficient to cancel the
aging program, thereby prolonging the reproductive period of the individual, i.e.,
increasing the possible total number of its descendants. In other words, the impact
of dietary restriction on lifespan is only indirectly related to ROSit is a regulatory effect, being first of all biology, and not chemistry. That is why such evident
signaling effects as short-term fasting (or, vice versa, smell of food), rather than
life-long food shortage (or excess) exert a powerful effect on the life-cycle
parameters. It is remarkable that a temporary dietary restriction (fasting) is better
than constant restriction. The signal can be given within a fairly short time,
whereas long-term starvation is clearly harmful for an organism.3
The signaling nature of the effect of dietary restriction provides a good rationale
for the experiments with methionine. Apparently, the organism determines the
amount of available food and, first of all, essential amino acids required for protein
synthesis, by monitoring the level of only one of themmethionine.
It seems quite significant that dietary restriction not only extends lifespan, but
also prolongs youth, as already mentioned by the discoverer of this phenomenon
C. M. McCay. In the food-restricted animals, age-related diseases are retarded and
Religious fasting probably prolongs life by short-term dietary restrictions. Believers are known
to live longer than atheists (Eng et al. 2002).
374
old animals become indistinguishable from young ones in behavior and even
exterior appearance. Quite illustrative in this respect are the recently reported
results of research of Weindruch and his group (Colman et al. 2009) on primates.
20-year-long experiments on 76 macaques (adult animals from 7 to 14 years old)
showed that a long-term 30 % dietary restriction gives the following effects: (1)
sharp decrease in age-related death rate (for over 30-year-old animals, 20 % in the
food-restricted group against 50 % in the control group fed ad libitum);
(2) absence of diabetes from the causes of death; (3) halving the death rate from
cancer (in macaques, this is primarily intestine adenocarcinoma); (4) decrease in
death from cardiovascular diseases; (5) decrease in osteoporosis; (6) arrest of the
development of such age-related traits as sarcopenia, decline in brain gray matter,
alopecia, canities, etc. By the age of 30 years, 80 % of the surviving control
macaques showed some traits of aging, whereas in the experimental group such
traits were observed in as few as 20 % of the animals.
The experiment on monkeys is still far from completion, and, therefore, we can
say nothing about the effect of dietary restriction on the maximum lifespan of
primates. However, some evidence on this subject is available for rodents
(MacCay et al. 1943; Stuchlikova et al. 1975). This evidence shows that the
median lifespan (50 % death rate) in mice and hamsters increases much more
markedly than the maximum lifespan. The simplest explanation of the mechanism
of this phenomenon is an arrest (or a strong retardation) of the aging program.
Therewith, other ontogenetic programs, first of all body growth, can also be
retarded. These phenomena are observed with a rather strong and long-term fasting
(MacCay and Crowell 1934). However, a more moderate dietary restriction can
prolong life while not causing inhibition of growth (Berg and Simms 1960).
The effects of dietary restriction and SkQ antioxidants have much in common.
Rectangularization of survival curves and decrease in early mortality rates are
observed with either treatment and the median lifespan increases much more than
the maximal lifespan (Anisimov et al. 2008, 2011; Skulachev et al. 2009). Both of
these factors operate not only, and not so much, as to prolong life as such, as to
prolong a healthy young life. Both are very effective in living beings quite different
in their systematic position (dietary restriction works well in yeast, insects, and
mammals, and SkQ, in fungi, crustaceans, insects, fish, and mammals). The effect
of both factors is clearly pleiotropic in nature, i.e., they influence a variety of
physiological functions, and the lists of those functions are similar. Cardiovascular
diseases, osteoporosis, vision disorders, and certain types of cancer are decreased,
graying and loss of hair as well as age-related depression (torpor) do not occur.
Contradictory data concerning effects of dietary restriction on sarcopenia and
immune responses have been reported. Some authors state that such exposure
adversely affects both the muscle system and immunity.4 On the other hand,
4
Fernandes et al. (Sun et al. 2001) noted that dietary restriction, especially at a young age,
inhibits interleukin secretion by macrophages as a response to bacterial lipopolysaccharides.
According to Gardner (2005), partial starvation reduces resistance to influenza virus.
16.2
375
Weindruch et al. (Colman et al. 2009) reported the lack of sarcopenia in monkeys,
and McCay et al. (MacCay and Crowell 1934), resistance to pulmonary diseases in
rats subjected to dietary restriction (cf. decrease in age-dependent involution of the
thymus and follicular spleen compartments OXYS in rats treated with SkQ1
(Anisimov et al. 2008; Skulachev et al. 2009; Obukhova et al. 2009)). At the same
time, the effects of dietary restriction and SkQ1 on wound healing are opposite:
starvation inhibited, while the antioxidant SkQ1 stimulated healing (Demianenko
et al. 2010). Dietary restriction decreases body temperature and inhibits growth,
which was not observed with SkQ1 (Skulachev et al. 2011). These data rule out
such a trivial interpretation of our data as the assumption that SkQ1 decreases
animal food intake, say, by decreasing appetite. Direct measurements on mice
treated with SkQ1 revealed no decrease in food intake at the low SkQ1 concentrations used in our experiments.
The fact that dietary restriction adversely affects some vitally important
parameters is not surprising. In fact, animals do not tend to overeat even if they are
not restricted in food. Usually, they eat as much as it is needed for their organism.
Therefore, long-term dietary restriction entails some disorders in vital functions. It
is also clear that such disorders are more probable the longer starvation continues.
We already mentioned that long-term and continuous dietary restriction is not
necessary for the geroprotecting effect. This explains the controversy in data on the
effects of dietary restriction. In cases when dietary restriction was not too severe
and not too long, positive effects were observed, whereas when gerontologists
overdid the restriction, unfavorable influence occurred. Thus, it is commonly
accepted that long-term dietary restriction decreases the frequency of estrous
cycles (sometimes until they cease completely) (Hopkin 2003), but already in 1949
Carr et al. (1949) showed the temporary dietary restriction with its subsequent
cancelation prolonged estrous cycles, which could be observed until extreme old
age. Let us remember that the same effect was found with SkQ1 (Anisimov et al.
2008, 2011; Skulachev et al. 2009). In principle, there is no need to starve through
the whole life if starvation is a signal to switch off the aging program. However,
there is a probability that too weak or delayed dietary restriction will only partially
retard the program, and the geroprotective effect will be weak. The use of SkQ1
appears not to pose such a threat, since SkQ1 acts at such low concentrations that
no adverse effects have been observed so far.
Another circumstance should be taken into account when considering restriction in eating as a geroprotector for humans. Actually, if dietary restriction is a
signal to warn against starvation, then the organism should respond not only by
prolonging life to compensate for the decay of birth rates in lean years. Other
responses also seem quite reasonable, and some of them are not as attractive as an
increase in a healthy life. For example, it was noted that a hungry mouse, once
finding itself in a squirrel wheel, does not want to leave it and runs from 6 to 8 km
overnight (with normal feeding, this distance is always shorter than 1 km) (Hopkin
2003). Obviously, this effect cannot be explained by starvation-induced exhaustion
and muscle weakness. More likely, we deal here with another response to the
starvation signalextreme anxiety and attempt to scan as large of territory as
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16.3
377
offspring with a changed genome, should be a signal for the organisms selfelimination, i.e., acute phenoptosis.
It seems quite possible that the mechanism of acute phenoptosis, like that of
slow phenoptosis (aging), is mediated by intramitochondrial ROS at early stages of
the process. If this assumption is valid, then the positive effect of SkQs not only on
aging, but also on a variety of acute pathologies both in young and old organisms
can be explained in terms of the neutralization of these ROS.
Thus, there is a chance to use SkQs as a tool in the rise of the machines, an
attempt of Homo sapiens to overcome genome tyranny and to cancel those
genome-dictated programs that are useful for genome evolution, but unfavorable
for an individual. Their cancelation would symbolize human conversion into
Homo sapiens liberatus,5 which would be the highest achievement of medicine in
the twenty-first century (Skulachev 2009a).
16.4 Conclusions
To prevent age-dependent increase in mitochondrial reactive oxygen species
(mROS), cationic derivatives of plastoquinone (PQ, a photosynthetic electron
carrier and very active antioxidant) were synthesized in our group. In the majority
of experiments, the compound called SkQ1 was used. It is composed of PQ
conjugated with decyltriphenylphosphonium cation that easily penetrates through
membranes due to delocalization of its positive charge over the bulky hydrophobic
phenyl residues. The following properties of SkQ1 were revealed. (1) SkQ1 has
low solubility in either water or hydrocarbons, but it is highly soluble in octanol
(1:10,000 water/octanol distribution coefficient) that means it has extremely high
affinity for membrane structures. (2) Experiments on bilayer planar phospholipid
membrane showed that SkQ1 is good penetrant for such a membrane. (3) SkQ1
accumulates in mitochondria in vitro in a DW-dependent manner. (4) In various in
vitro systems, SkQ1 is a very potent antioxidant. (5) In mitochondria, SkQ1 is also
a prooxidant if added in micromolar concentrations. However, these concentrations are very much higher than those required for the antioxidant effect of SkQ1,
which is quite measurable at nanomolar concentrations. (6) In respiring mitochondria, SkQ1 operates as a rechargeable antioxidant that is reduced at center
i (heme bH) of respiratory chain complex III. (7) In human cell cultures, SkQR1, a
fluorescing SkQ1 analog, was shown to stain all the mitochondria and only the
mitochondria, which can be easily explained by the fact that the mitochondrial
interior is the only negatively charged intracellular compartment. (8) In the same
cell cultures, SkQ1 and SkQR1 are very effective inhibitors of H2O2-induced
apoptosis; the SkQ1 analog lacking PQ is ineffective. (9) In vivo treatment with
SkQ1 increased the median lifespan of some tested fungi, crustaceans, insects, fish,
378
and mammals. (10) In animals, appearance of typical traits of aging were retarded,
stopped, or even reversed by SkQ1. These traits included osteoporosis, lordokyphosis, achromotrichia, balding, slow wound healing, involution of thymus and
spleen follicles, sarcopenia, increase in left ventricle volume of the heart, retinopathy, glaucoma, cataract, dry-eye syndrome, disappearance of estrous cycles in
females and libido in males, age-dependant changes in behavior, etc. In the cases
of age-related eye diseases, drops of very diluted (250 nM) SkQ1 solution were
efficient in rats, rabbits, dogs, cats, and horses. (11) Double-blind clinical trials on
humans suffering from dry-eye syndrome clearly showed that drops of SkQ1
effectively treated this age-related disease regarded as incurable. In 2012, these
drops became available in drugstores in Russia. (12) A single injection of SkQ1
was shown to prevent the death of animals after a crisis such as kidney ischemia,
rhabdomyolysis, pyelonephritis, etc. It decreases infarct area in heart and brain
after experimental ischemia in rats. It was concluded that SkQ1 is competent in
inhibiting a programmed death of organisms (phenoptosis), being effective in
both slow (aging) and fast (sudden death after crisis) version of this phenomenon
which is counter-productive for the individual but apparently favorable for evolution of the species. (13) The majority of the effects of SkQ1 are similar to those
of partial food restriction. This indicates that food restriction, like SkQ1 treatment,
is a way to downregulate the aging program. However, in contrast to SkQ1, food
restriction has undesirable side effects revealed in rodents, such as decrease in
fecundity, elevated anxiety, and a tendency to scan as large area as possible.
Apparently, food restriction stimulates a forager activity of the individual, being a
signal of starvation, and downregulation of the aging program prevents the agelinked decrease in efficiency of functioning of the organism under adverse conditions. (14) It is hypothesized that SkQ-type treatments might be used to switch
off the aging program as well as some other programs counter-productive for an
individual but useful for evolution.
Acknowledgments The authors would like to express their gratitude to their families for
patience and support expressed over several years of work on this book. Excellent work of Drs.
Anna Brzyska, Richard Lozier, Konstantin Lyamzaev, and Antonina Pustovidko in preparing the
English version of the book is greatly appreciated. The original illustrations and the general
graphic style of the book were created by one of the authors (F.O.K.) in LLC MASTERMULTIMEDIA (www.master-multimedia.ru).
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Appendices
Appendix 1
Energy, Work, and Laws
of Thermodynamics
A.1 Introduction
Energy (from Greek eme9qceia1action, activity) can be generally defined as the
property of a material system that characterizes the ability of this system to
produce some changes in the environment (work, W). Such a work can consist of
growth of the total amount of matter in a system, the uneven distribution of matter
particles in space, and/or movement of these particles. Physical interactions
between matter particles in a system are accompanied by the mutual
transformation of different types of energy (mechanical, thermal,
electromagnetic, gravitational, nuclear, and chemical). How is life ensured by
energy? The laws of thermodynamics provide us with a quantitative answer to this
question. Energy transductions in living self-replicating material systems
(organisms) follow the laws of thermodynamics. It seems quite remarkable that
the first law of thermodynamics (the law of conservation and transformation of
energy) was first formulated in 1841 by the German physicist and physician Julius
Robert von Mayer as the result of his studies of the energy processes in the human
organism; it was only some time later that this law was applied to the systems of
technical energetics.
Thermodynamics deals with three types of systems: isolated, closed, and open.
Hypothetical isolated (adiabatic) systems are completely self-contained, i.e., there
is no exchange of matter, energy, and information with the environment. Closed
systems are materially self-sufficient, but they are open energetically and
informationally. In other words, there is exchange of energy and information,
but not matter through the borders of closed systems. Many nonliving systems of
technical energetics function in this way. Living organisms are open systems that
constantly exchange matter, energy, and information with the environment.
The term heat (Q) is used in thermodynamics as the measure of energy passing
from one body to another. A system uses received energy to perform an external
383
384
work and also to carry out transitions between the alternative states of the
components of the system. Changes in the amount of work can be calculated as the
product of pressure (P) and an infinitesimal increment of volume (DV):
DW PDV
A1:1
A1:2
385
can be viewed as closed systems, which means that the equations of equilibrium
thermodynamics can be applied to these processes. Thus, we conclude (on the
basis of these equations) that for any given system the potential ability to perform
work is determined by the degree of deviation of its components from the
equilibrium state. To use in practice the approximate quantitative estimate of open
systems, one should understand the nature of the equilibrium state of a particular
chemical reaction and estimate equilibrium shift of the real reaction with respect to
the equilibrium state predicted by the equations of classical equilibrium
thermodynamics. It is this shift in equilibrium that determines the quantitative
ability of the reaction to support performance useful work. Furthermore, the
mathematical apparatus of equilibrium thermodynamics can be effectively used for
the identification of forbidden reactions. Only processes leading to increase in
entropy and reduction in energy reserve can proceed spontaneously (without any
energy input into the system).
The internal energy of a system (U) is defined as the total energy of the given
system minus the kinetic energy of the system as a whole (energy of motion in the
environment) and the potential energy of the system in external force fields (when
386
A1:3
A1:4
A1:6
387
A1:8
A1:9
A1:10
The type of Gibbs energy change makes it possible to estimate the maximal
theoretical possibility of a process. When DG \ 0, a process can proceed
spontaneously if no kinetic factors interfere with it. For instance, a negative value
of Gibbs energy change does not hinder reduction of O2 to 2H2O by NADH with
no mediators involved. However, an NADH molecule, when oxidized, releases
two electrons while an oxygen molecule, when reduced to water, accepts four
electrons. As a result, such a process is forbidden because of its not matching the
Schaeffer parity rule, which presumes the equal number of electrons released and
accepts in one and the same reaction. Respiratory chain electron carriers mediating
electron transfer from NADH to O2 help to outflank kinetic limitations of the
thermodynamically possible process of NADH oxidation by oxygen. When
DG = 0, the system is in the state of chemical equilibrium, and hence no gross
transformations of the system components occur. When DG [ 0, a process can
proceed only if there is an input of energy from the outside. For instance,
formation of glucose from water and carbon dioxide in photosynthesis is coupled
to Gibbs energy increase, and it can proceed only due to the input of the energy of
photons into the system. Change in Gibbs energy during biochemical reactions can
be determined in several ways: on the basis of affinity constant, values of standard
redox potentials, calorimetric measurement of heat produced in a reaction, and
also by summing standard values DH0 and TDS0 of the formation of the reaction
components from the respective simple elements (the latter are known from
tables).
388
A1:11
A1:12
A1:13
389
390
391
392
Appendix 2
Prosthetic Groups and Cofactors
Figs. A2.1, A2.2, A2.3, A2.4, A2.5, A2.6, A2.7, A2.8, A2.9, A2.10, A2.11, A2.12,
A2.13
393
394
Fig. A2.1 Structural formulas of various hemes of the enzymes of electron transport chains
395
Fig. A2.2 Chlorophylls of photosynthetic electron transport chains of prokaryotes and plants
396
Fig. A2.3 Quinone derivatives participating in enzymatic redox processes: the
number of isoprene residues
(n) varies in different species
from 6 to 10; ubiquinones
have been found in bacterial
and mitochondrial membranes, menaquinones in
bacterial membranes, plastoquinone in the membranes
of chloroplast thylakoids and
cyanobacteria, and rhodoquinone in the respiratory chain
of Ascaris imago
397
398
Fig. A2.7 Structural formulas of thiamine pyrophosphate, dihydrolipoic acid, and coenzyme A
399
400
401
Fig. A2.11 Structural formulas of different versions of coenzyme F420 (methanogenic archaea)
Fig. A2.12 Methanophenazine and dihydromethanophenazine of methanogenic
archaea
402
Fig. A2.13 Structural formulas of methanofuran
(MFR), formylmethanofuran,
and carboxymethanofuran
(methanogenic archaea)
Appendix 3
Inhibitors of Oxidative Phosphorylation
403
404
405
Appendix 4
Plant Hormones
Fig. A4.1
407
Appendix 5
Mitochondria-Targeted Antioxidants
and Related Penetrating Compounds
Fig. A5.1
409
410
411
Appendix 6
Mitochondria-Targeted Natural Rechargeable
Antioxidant
413
414
and methionine is transformed into the stable and harmless methionine sulfoxide
(in contrast, another sulfur-containing amino acid, cysteine, when interacting with
ROS, forms a very aggressive radical1). In addition, it seems to be very important
that methionine sulfoxide can be reduced by electrons from the respiratory chain,
this leading to its regeneration to the original methionine:
NADPH ? thioredoxin reductase ? thioredoxin ? methionine sulfoxide
reductase ? methionine sulfoxide
Thus, methionine of the proteins encoded by mitochondrial DNA is an analog
of SkQs, for it is also a rechargeable antioxidant located in the inner mitochondrial
membrane (Fig. A6.2). It seems quite significant that additional methionines
resulting from changes in the mitochondrial genetic code are located on the
periphery of the protein molecule, for they meet ROS on their way toward the
active sites of the key proteins of oxidative phosphorylation. This location of
methionine residues originates from the fact that the same sites were formerly
occupied by the residues of the hydrophobic amino acid isoleucine. The discussed
proteins are integrated into the membrane as bricks into the wall, and surface
hydrophobic amino acids strengthen their bonds with the intramembrane partners.
The antioxidant role of methionine residues has been recently confirmed by Luo
and Levine in experiments on E. coli (Luo and Levine 2009). The bacterium was
grown in the medium containing no methionine (it was replaced by norleucine).
Because the protein biosynthesis system of E. coli can use norleucine instead of
methionine, one could observe gradual accumulation of cells with methionine
being replaced by norleucine in their proteins. When the level of replacement
reached 40 %, the cells were washed from free norleucine and methionine was
added. As a result, the amount of free methionine and S-adenosylmethionine in E.
coli cells became normal, while the amount of methionine in proteins remained
reduced. These cells were found to be much more sensitive to toxic effect of H2O2
than the control cells (Fig. A6.1).
It is not surprising that there exists an inverse correlation between the organism lifespan and
the number of cysteine residues in proteins encoded in mitochondria (Moosmann 2011).
415
Fig. A6.2 SkQ and methionine as rechargeable antioxidants protecting mitochondria from ROS.
ROS, reactive oxygen species; IOS, inactive oxygen species; SkQH2, reduced form of SkQ;
3?
SkQ-, anion-radical of SkQ; b2?
H , reduced cytochrome bH; bH , oxidized cytochrome bH;
methionine and methionine sulfoxide, respective amino acid residues in proteins encoded by
mitochondrial DNA; Trxr, reduced thioredoxin; and Trxo, oxidized thioredoxin. Respiration
3?
regenerates b2?
H and Trxr by reducing bH and Trxo
Appendix 7
Key Participants of the Project Practical
Application of Penetrating Cations
Fig. A.7.1
417
418
Fig. A.7.1 Some key participants of the project Practical application of penetrating cations
(V. P. Skulachev, Chairman of Scientific Board, Moscow)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
(25)
(26)
(27)
(28)
(29)
(30)
419
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Author Index
A
Abdulaev, N., 141
Adler, J., 196, 197
Agre, P., 5
Aleem, M., 130
Anisimov, V., 338, 356, 358, 360, 361, 375
Antonenko, Yu., 346, 356358, 363, 366
Archakov, A., 264
Asadov, A., 43
Austad, S., 326, 330, 334336, 343, 372
B
Bakeeva, L., 256, 264, 356, 364, 365
Baltscheffsky, M., 184, 185
Barja, G., 340
Barquera, B., 279
Bate Smith, E., 84
Baykov, A., 185, 285
Belitser, V., 3, 174
Belogurov, G., 285
Berg, H., 197, 200, 201, 311, 374
Bibikov, S., 152, 153, 251
Blobel, G., 417
Blumenfeld, L., 16
Bogomolni, R., 145, 151
Bonduryansky, R., 327
Boyer, P., 5, 167, 169, 172
Brand, M., 340
Brassil, C., 327
Bremer, B., 215, 228
Brenner, S., 318
Brodie, A., 246, 287
Brown, I., 267, 269
Bubenzer, H., 256
Budrene, E., 311, 313
Burton, M., 255
C
Cannon, B., 236, 358, 364
Carr, C., 373
Chance, B., 103, 108
Chen, L., 249, 258, 315
Chentsov, Y., 256
Cherepanov, D., 417
Chernyak, B., 317, 324, 358
Chikunov, A., 355
Chubays, A., 355
Ciechanover, A., 417
Cleveland, L., 204
Cohen, G., 218
Comfort, A., 326, 328, 342, 372
Crick, F., 4
D
Dalton, H., 128
van Dam, K., 4
Danielson, L., 187
Darwin, C., 327, 334, 376
Dawkins, R., 329, 376
Deisenhofer, J., 5, 3739, 41, 42, 45
Deripaska, O., 355
Dibrov, P., 289
Dilman, V., 338, 342
Dimroth, P., 275, 279, 291, 292
Donehower, L., 343
Drachev, L., 44, 45
Drachev, V., 260
Drize, N., 358
E
Egorov, M., 417
Eisenbacher, M., 197
423
424
E (cont.)
Emanuel, N., 339
Engelhardt, V., 3, 87, 245
Epstein, W., 209, 301
Erichev, V., 364
Ernster, L., 4, 187
F
Feldman, R., 169
Feniouk, B., 417
Fernandes, J., 374
Filenko, O., 358
Finch, C., 338, 342
Foissner, I., 259
Fritz, I., 9, 214, 215
G
Galen, C., 318
Gardner, E., 374
Garrahan, P., 291
Gauthier, G., 256
Glynn, I., 291
Goglia, F., 235, 239
Goldsmith, T., 334
Goldstein, N., 326
Green, D., 89, 307, 345
Greenhut, S., 265
Grinius, L., 228
Gulevitch, V., 82, 214
H
Haldane, J. B. S., 3
Hamilton, W., 376
Harman, D., 339, 372
Harold, F., 268, 282
Heefner, D., 282
Hekimi, S., 337, 338
van Helmont, J. B., 3
Henderson, R., 39, 141, 143
Holzenberger, M., 338
Horwitz, H., 232, 236, 318
Huber, R., 5, 37, 38
I
Iomdina, E., 364
J
Jasaitis, A., 139, 140
Johansson, B., 232
Author Index
K
Kapelko, V., 364
Kaulen, A., 45, 145, 146
Kauppinen, R., 249
Keilin, D., 13
Kenyon, C., 337
Kerr, J., 318
Khorana, H., 141
Klingenberg, M., 221, 233, 234
Kolosova, N., 358, 361, 362
Kopenkin, Ye., 363
Kopnin, B., 365
Korshunova, G., 312, 314, 356, 366
Krasnovsky, A., 32
Krebs, H., 4, 73
Krimberg, R., 214
Kroemer, G., 5, 320
L
Labedan, B., 228
Lahti, R., 285
Laimins, L., 209, 301
Lamarck, J.-B., 240
Larsson, N., 334
Lewis, K., 152, 196, 200, 322, 329
Liberman, E., 187, 345
Lichtenstein, A., 322
Lipmann, F., 9
Loison, A., 327
Longo, V., 305, 306, 315, 338
Lozier, R., 145, 146, 378
Lyamzaev, K., 317, 356, 376, 378
M
MacKinnon, R., 5
Macnab, R., 288
Malinen, A., 285
Manskikh, V., 322
Maslov, S., 87, 237, 238, 246
McCay, C., 372, 373, 375
Medawar, P., 327
Meisel, M., 260
Michel, H., 5, 37, 112
Mikhelson, V., 361
Mitchell, P., 4, 5, 10, 48, 51, 98, 106, 215, 219,
265
Mokhova, E., 238
Moore, J., 255
Moosmann, B.
Mozenok, E., 203
Mulkidjanyan, A., 293, 300
Murphy, M., 345, 346
Author Index
N
Nedergaard, J., 236
Newmeyer, D., 5, 319
O
Obukhova, L., 364, 369, 375
Oesterhelt, D., 139, 140, 144
Okunuki, K., 245
Ovchinnikov, Y., 141, 142, 153
P
Pande, S., 216
Papa, S., 98
Park, T., 341
Parvin, R., 216
Pasyukova, Ye., 358
Pelicci, P., 337
Pfenig, N., 292
Pfenninger-Li, X., 279
Ponnamperuma, C., 16, 17
Postgate, J., 128
Prolla, T., 339
Proverbio, F., 284
R
Racker, E., 161, 162
Ramsay, R., 216
Reznik, D., 335, 336
Rich, P., 36, 49, 51, 52, 107, 277, 278
Rickenberg, H., 218
Robertson, T., 372
Roginsky, V., 355, 356
Roseman, M., 265
Rosen, B., 300
Rothwell, N., 236
Rottenberg, H., 221
Ruuge, E., 417
Ryazanov, A., 364
S
Sadovnichii, V., 417
Sagan, C., 16
Sazanov, L., 93, 94, 96, 98, 99, 101,
102, 190
Schink, B., 133, 186, 292, 297
Scrable, H., 343
Semenov, A., 45
Senin, I., 364
Severin, F., 305, 323, 340, 356, 366
Severin, S., 8284, 345
425
Severina, I., 253, 254, 357, 366
Shidlovsky, K., 358
Shuvalov, V., 43
Sigman, D., 169
Skou, J., 5, 283
Skulachev, M., 356, 366, 376
Slater, E., 4, 102, 104
Smith, R., 232, 235, 255, 345, 346
Sohal, R., 340
Sommer, S., 322
Sotnikova, L., 363
Spirin, A., 18
Spivak, I., 361
Starkov, A., 312
Stock, M., 163166, 172, 174, 236
Stoeckenius, W., 139, 140, 144, 145
Stuchlikova, E., 373, 374
Sulston, J., 318
Sumper, M., 263
Szent-Gyrgyi, A., 4, 5, 17
T
Tanaka, K., 260
Teather, R., 219
Temkin, M., 16
Tikhonova, G., 130
Tokuda, H., 277, 279
Trauble, H., 263
Trissl, H., 47
Tubbs, P., 216
U
Unemoto, T., 277, 279, 287
Unwin, N., 141, 143
V
Verkhovsky, M., 115, 116, 279, 280
Vinci Leonardo da, 3
Vinogradov, A., 27, 96, 168
Vysokikh, M., 346, 357, 358, 366
W
Walker, J., 5, 160, 163
Wallace, R., 327, 328, 329, 376
Wang, X., 5, 319
Waterbury, J., 203
Watson, J., 4
Weindruch, R., 374, 375
Weismann, A., 327329, 331, 332, 376
West, I., 219, 266
426
W (cont.)
Wikstrm, M., 110, 115, 116
Woldegiorgis, G., 224
Y
Yaguzhinsky, L., 345
Yoshida, M., 172, 173
Yoshikawa, S., 112
Author Index
Z
Zamyatnin, A., 419
Zassenhaus, H., 339
Zinovkin, R., 362, 363
Zorov, D., 258, 260, 262, 315
Subject Index
A
Acetyl-CoA, 74, 75, 214, 276
Acetyl phosphate, 77
Acidophilic bacteria, 34
Aconitase, 312, 313, 315, 316, 323
Adenylate cyclase, 233
Adenosine diphosphate (ADP), 168170, 172,
174, 175
inhibition of FoF1-ATPase, 180
Adenosine monophosphate (AMP), 15, 16,
183, 184
Adenosine triphosphatase (ATPase)
Ca2?, 27, 180, 301
Cl-, 301, 302
classification, 176
type E1E2, 180
type FoF1, 177
type V0V1, 179
H?, 2123, 152, 176185, 212, 221, 251,
268, 269, 288, 297, 301
in anaerobic bacteria, 177, 182
in plasmalemma, 183
K?, 5, 182, 209, 301
Na?, 5, 282, 283, 288, 293
Na?/K?, 27, 154, 180, 283, 284, 301
Adenosine triphosphate (ATP), 14, 15
as convertible energetic currency, 11, 15,
22, 185, 252, 265, 301
Adenosine triphosphate synthase (ATPsynthase)
in chloroplasts, 159, 161, 183, 268
mechanism, 172, 198, 221, 226, 268
mitochondrial, 160, 161, 163, 166
Na?, 27, 293
stoichiometry H?/ATP, 165, 174176, 180,
186
3D structure, 162164
B
Bacteriochlorophyll, 32, 3437, 39, 41, 42, 44,
48, 54
dimer, 34, 35, 38, 42, 44, 53
Bacteriopheophytin, 34, 35, 37, 42, 44
Bacteriorhodopsin
3D structure, 142
conformational changes, 143, 148, 149
dark adaptation, 145
electrogenic stages, 146
427
428
B (cont.)
generation of D
lH?, 139, 141
in attractant response, 152
photocycle, 143145, 147150, 153
quantum yield, 23
schiff base, 140144, 147150
two-dimensional crystals, 139, 143
Bilayer membrane, 32, 88, 222, 227, 299,
357, 377
Binuclear center, 110, 113, 114, 116, 121, 123,
125, 126
Bioenergetics
definition, 3
evolutional aspects, 1315
relation to other biological
sciences, 57, 18
Biotin, 173, 275
Bongkrekic acid, 221, 224
British anti-lewisite, 108
Brown fat, 232234, 236239, 246
Buffers of D
l H?, 11, 265
C
Calcium ion
accumulation in mitochondria, 208, 211
activation of phospholipase A2, 250
as secondary messenger, 250
Capsaicin, 96, 97, 341
Carboxyatractyloside, 221, 224, 238
Cardiolipin, 362, 365, 366, 369
Carnitine, 214217
Carotenoids, 33, 43, 140
Caspase, 319
Catalase, 248, 341
Chemiosmotic theory, 5
Chloride transport, 67
m-Chlorocarbonyl cyanide phenylhydrazone
(CCCP), 290
Chlorophyll, 2225, 31, 32, 54, 5658, 62, 63,
87, 151, 252, 253
Chlorophyll dimer, 56, 61, 63
Chloroplast, 55, 58, 62, 66, 132, 253
buffering of D
l H?, 67
outer membrane, 204
photoredox chain, 52, 53, 55, 57,
67, 151
rotation, 203, 204
structure, 56
Cholesterol, 204, 264
Chromaffin granules, 212
Chromatophore, 23, 32, 35, 45, 47, 5153,
106, 169, 184187, 190,
268, 269
Subject Index
Citrate, 186, 213, 275, 276, 287, 313
Cobalamine (vitamin B12), 280, 281
Coenzyme A (CoA), 17, 18, 84
Coenzyme B (CoB), 135
Coenzyme F420, 96, 135137, 401
Coenzyme M (CoM), 134
Coenzyme Q (CoQ)
in reaction center complex, 42, 43, 45, 48,
49, 51
in respiratory chain, 90, 92, 102, 104, 123,
130, 190
Complex bc1. See Complex III
Complex b6 f, 5658, 60, 63, 64, 66, 105,
108, 131
Complex I, 9297, 308
3D structure, 99, 101, 102
bacterial, 9395
electron transport, 99
generation of D
l H?, 97
inhibitors, 96, 97, 341
mitochondrial, 99, 102
redox centers, 90, 94
stoichiometry H?/e, 98, 99
subunit composition, 9396
Complex II. See Succinate
dehydrogenase
Complex III (CoQH2:cytochrome
c- oxidoreductase)
3D structure, 103, 104
in bacterial photoredox chain, 106
in respiratory chain, 104
inhibitors, 108, 312
redox centers, 104
subunit composition, 104, 105
Complex IV. See Cytochrome c oxidase
Copper, 24, 5659, 85, 90, 98,
110115, 121, 123, 125, 126
Coupling ion, 27, 119, 285, 297, 300
Creatine phosphate, 14, 108, 217
Cyanide, 121, 125, 131, 132, 152, 240,
245247
Cyanobacteria, 55, 131
Cyclic adenosine monophosphate
(cAMP), 233, 251
Cyclic guanosine monophosphate
(cGMP), 154
Cyclosis, 203, 204
Cytochrome
a, 109
b, 35, 49, 51, 56, 63, 64, 90, 103106, 136,
323, 339
b5, 246, 264, 265
b559, 61, 62
c, 109
Subject Index
c1, 49, 51, 52, 90, 102106, 108, 109
d, 109
definition, 24
f, 56, 59, 6365
o, 52
P-450, 246, 247
tetraheme c-type, 3741, 44, 47, 49, 51
Cytochrome c oxidase, 88, 106, 108, 110, 113,
115, 120, 123, 125, 128, 131, 337
3D structure, 112
aa3-type, 123, 125
cbb3-type, 123, 128
generation of D
lH?, 115
inhibitors, 116
redox centers, 110
stoichiometry H?/e, 115, 116
subunit composition, 110112
D
Decarboxylase
glutaconyl-CoA, 276
methylmalonyl-CoA, 276, 277, 292
Na?-motive, 275
oxaloacetate, 275, 276
Deoxyribonucleic acid (DNA), 18
Devescovinid, 204, 205
N,N-Dicyclohexylcarbodiimide (DCCD), 152,
167, 171, 177, 180, 181, 202, 203
Diethylstilbestrol, 167, 180, 181
2,4-p-Dinitrophenol, 238, 259
Diuron, 60, 259
E
Electron magnetic resonance (EPR), 94, 95
Endoplasmic reticulum, 6, 27, 178, 183, 246,
264, 301, 308
Endosome, 178, 179, 183
N-Ethylmaleimide, 180, 282
F
Factor Fo
inhibition by DCCD, 171
sensitivity to oligomycin, 171
structure, 161
subunit composition, 160, 161, 165,
171, 198
Factor F1
3D structure, 162, 164
ATP hydrolysis, 167169
catalytic centers, 167, 168, 177
inhibition by ADP, 169
429
nucleotides binding, 167, 168, 172, 179
subunit composition, 177
synthesis of bound ATP, 169
unisite catalysis, 167
Factor F6, 161
Fatty acid
as uncouplers in thermogenesis, 234
covalent binding to proteins, 38, 234
dicarboxylic, 201, 233
b-oxidation, 91, 216, 217, 233, 235
transport, 214, 217, 263, 345
Fatty acylcarnitine, 214216
Fatty acyl-CoA, 91, 215, 233, 263
Fentons reaction, 325
Fermentation, 3, 20, 7982, 132134,
182, 276
Ferredoxin, 53, 57, 5860, 66, 96, 278
Ferredoxin-NADP? reductase (FNR), 60, 66
Ferricyanide, 95
Flagellin, 197, 198
Flagellum, 195201, 204, 205, 290, 291
bacterial, 195197, 205
eukaryotic, 195, 205
Flavin adenine dinucleotide (FAD), 18, 54, 58,
120, 125, 246, 278280
Flavin mononucleotide (FMN), 90, 94, 95, 99,
101, 278280
Formate, 81, 119, 132134, 276,
281, 293
Formate dehydrogenase, 81, 281, 293
Free respiration, 231, 232, 240, 245248
Fumarate, 75, 119, 126128, 312
Fumarate reductase, 126, 127
G
Generator of D
lH?e
primary, 287
secondary, 176179, 181, 282, 283
stoichiometry H?/e, 98, 115, 116,
124, 125
Glaucoma, 360, 364, 371, 378
Glucose, 25, 26, 71, 7375, 80, 81, 119, 127,
133, 184, 212, 302
Glutathione, 309, 341, 366
Glyceraldehyde phosphate
dehydrogenase, 76
Glycerol, 38, 40, 71, 233, 248, 250
Glycolysis, 19, 21
Gramicidin, 218
Green photosynthetic bacteria, 22, 53
Guanosine diphosphate (GDP), 77, 154, 233
Guanosine triphosphate (GTP), 72, 77, 154,
221, 222, 233
430
H
Halophilic bacteria, 288, 297, 300
Halorhodopsin, 149, 150, 152, 153,
299, 301
Heme
bH, 35, 36, 49, 64, 106, 312, 377
bL, 4951, 64, 106, 312
chlorin, 125
high-spin, 111, 116, 125
low-spin, 111, 116, 125
Hemoglobin, 111
2-Heptyl-4-hydroxyquinoline
N-oxide (HQNO), 66, 280
Hexokinase, 73, 250
Hibernation, 236
Hydrogenase, 96, 136, 137, 276
Hydroxylamine, 167, 180, 181
Hypoxia, 324326
K
a-Ketoglutarate, 11, 75, 77, 78, 122,
222, 249
Krebs cycle, 7375, 77, 91, 127, 128, 186,
190, 213, 250, 312, 323
L
b-Lactamase, 227
Lactate, 20, 80, 81, 83, 84, 119, 132, 212,
246, 276
Leader peptidase, 225
Lipase, 233
Lipoic acid, 77
Lipophilic ions, 136
Lipoprotein, 198
Liposome, 149, 219, 357
Lysophospholipids, 250
Lysosome, 6, 178, 179, 183, 317
M
Macroergic bond, 14
Malate, 119, 127, 128, 213, 240, 248, 249
Malateaspartateglutamate shuttle, 248
Malate dehydrogenase, 249
Melatonin, 342
Melibiose, 287
Melittin, 227
Membrane, 101, 149, 161, 180, 188, 227,
252, 300
Menadione (Vitamin R3), 247
Menaquinone (MQ), 40
Methane, 133135, 280, 281, 293, 294
Subject Index
Methanofuran, 281, 293
Methanogenesis, 134, 135
Methanophenazine, 136, 137
Methionine, 373
Methylamine, 134, 135
Methylmalonyl-CoA, 276, 277, 292
Mild uncoupling, 312, 367
Mitochondrion
biogenesis, 226
Ca2? transport, 27
filamentous, 254, 256, 258, 260
giant, 255
inner membrane, 211, 216, 232,
246, 251
junction, 256, 258, 260, 261
lateral transport, 261, 264
motility, 224
origin, 224, 226
outer membrane, 256, 260
shape, 254, 258
staining by rhodamine, 161, 249, 250,
255, 259
Mitoptosis, 315, 317, 322
Molybdenum, 124, 282
Monensin, 290
Motility, 195, 196, 202, 203, 205, 224, 254,
267, 289291
of bacteria, 197, 203
of protozoa, 203, 205
of spermatozoa, 195
Mucidin, 108
Myxothiazol, 108, 240, 312
N
Na?/K?-gradient as buffer
for D
lH?, 53, 265
?
Na -cycle, 297, 300
Na?-motive force(s), 10, 11
Na?-motor, 290, 291
NADH dehydrogenase, 96, 120, 278
Necrosis, 320323, 325, 328, 358, 360
Nicotinamide adenine dinucleotide
(NAD(H)), 73
Nicotinamide adenine dinucleotide
phosphate (NADP(H)), 18
Nigericin, 218, 221, 249
Nitrate
as inhibitor of V0V1-ATPase, 180
as terminal electron acceptor, 124, 127,
135
reductase, 124, 126
Nitrite, 92, 124, 131
p-Nitrophenol, 146
Subject Index
Nonheme FeS proteins, 24, 35
in complex I, 9496
in complex III, 104106
in noncyclic photoredox chain, 56
in photosystem I, 58, 59
Nonheme iron
in alternative oxidase, 121
in photosystem II
in reaction center complex
Noradrenaline, 233, 236, 238
Nuclear magnetic resonance
(NMR), 164, 165
Nucleoside diphosphate kinase, 222
Nucleus, 6, 33, 93, 104, 161, 255, 315, 345
Nystatin, 204
O
Obesity, 236
Oligomycin, 160, 167, 170, 171, 180,
185, 238
Organoptosis, 324, 328
Osmoregulation, 176, 181
Osmotic work, 11, 66, 182, 207, 213, 218, 287,
297, 300, 302
Osteoporosis, 330, 360, 369, 374, 378
Ouabain, 284, 301
Oxaloacetate, 249, 275, 276
P
Palmitoyl carnitine, 216
Peptidoglycan, 200
Peroxisome, 6, 248, 308, 316
o-Phenanthroline, 47, 62
Phenazine methosulfate, 60
Phenoptosis, 322324, 326, 328330, 333,
376, 377
Pheophytin, 55, 61
Phoborhodopsin, 151, 152
Phosphatidylinositol, 250
Phosphodiesterase, 154
Phosphoenolpyruvate, 74, 77, 78, 207, 302
3-Phosphoglyceraldehyde, 78
2-Phosphoglycerate, 77, 78
Phospholipase, 250
Photophosphorylation, 61
Photoreceptor, 151, 153, 154, 364
Photoredox chain
cyclic, 52, 57, 67, 151
in chloroplasts and cyanobacteria, 59,
299
in purple bacteria, 24, 34, 56
noncyclic, 5355, 57, 67, 151, 190
431
in chloroplasts and cyanobacteria, 59,
151
in green sulfur bacteria, 54, 55
Photosystem I
3D structure, 58
electron transport, 60
generation of D
l, 60, 61
localization, 67, 68
subunit composition, 58, 59, 61
Photosystem II
3D structure, 61, 62
electron transport, 62, 63
generation of D
l, 63
localization, 67, 68
subunit composition, 61, 62
Piericidin, 96
Plasmalemma, 6, 27, 179183, 207, 247,
302, 309
Plastocyanin, 51, 54, 5660, 65, 66,
108, 131
Plastoquinone (PQ), 24, 55, 57, 60, 108, 132,
309, 355, 366, 377
Potassium-transporting system, 209
Progeria, 329, 330, 362, 364
Propionate, 64, 65, 104, 132, 276, 292
Propionyl-CoA, 276, 292
Protein
binding to membrane, 38
inhibitor F1, 161
kinase, 233, 252, 320, 338
p53, 343, 365, 368
p66Shc, 337, 338, 341
Rieske, 51, 52, 90, 102106, 108
transport through membrane, 21, 227
Protein phosphatase, 252
Proteoliposome, 45, 47, 48, 60, 106, 107, 146,
181, 184, 186, 187, 219, 265
Protometer, 152, 153, 251
Proton motive force (Dp), 10, 88
Proton pump, 22, 9799, 102, 116, 123, 125,
126, 129, 139, 145, 184, 189
Protonophore, 186, 203, 235, 277, 282, 290,
297, 366
Purple membrane, 139141, 143,
146, 253
Purple photosynthetic bacteria, 52, 53
Pyrophosphate inorganic (PPi), 15, 159, 183,
184, 186, 268, 269
Pyrophosphate synthase, 52, 159, 183186
Pyruvate, 73, 74, 7781, 122, 127, 233,
275, 276
Pyruvate dehydrogenase
complex, 81
Pyruvate formate lyase, 81, 276
432
Q
Q-cycle, 51, 55, 56, 63, 66, 90, 96, 106,
299, 312
Quinol oxidase
ba3-type, 123
bd-type, 125, 126, 128, 129, 131
bo-type, 125, 126
Quinone, 277, 278
R
Ratio P/O, 174, 237, 238, 240, 246
Reaction center complex (bacterial)
3D structure, 37
electron transport, 60, 61
generation of D
l H?, 33, 101, 253
redox centers, 61, 63, 94, 110
subunit composition, 3637
Reactive oxygen species (ROS), vi, 101,
305, 377
Redox loop (Mitchell loop), 298, 299
Regulation of pH in cytoplasm, 288
Repellent, 151, 152, 196, 251
Respiratory chain, 87
Respiratory control, 122, 238, 312
Reticulum mitochondriale, 254, 259
Retinal, 149
Reverse electron transport, 11, 67, 130, 191,
312, 340, 341
Rhodamine, 249, 259
Rhodoquinone (RQ), 127
Rhodopsin
cGMP cascade, 154
generation of DW, 45, 48, 49, 297, 301
photocycle, 143145, 147150, 153
Riboflavin (Vitamin B2), 278, 279
Ribonucleic acid (RNA), 18, 183,
306, 309
messenger, 160, 233
ribosomal, 18
Ribozyme, 18
Rotenone, 96, 97, 240, 246, 259, 312
S
Sarcopenia, 330, 333, 370, 374, 375, 378
Secretory vesicles, 178, 179
Sensory rhodopsin, 151, 152
SkQ, 355, 357, 358, 362, 364368, 372, 374,
376, 378
Subject Index
Skulachev ion, 345
Special pair. See Bacteriochlorophyll dimer
Spermatozoid, 195, 255
Steroid hormones, 246
Stoichiometry H?/e, 98, 115, 116, 124126,
136, 137
Stoichiometry H?/ATP, 180, 186
Submitochondrial particles, 187
Succinate, 75, 91, 119, 126128, 132, 172,
174, 209, 211, 276, 292, 312
Succinate dehydrogenase, 91, 126, 312
Succinyl-CoA, 392
Sulfite, 169
Symporter
Cl-,H?, 150
H?,metabolite, 176, 288
H2PO4-, H?, 175, 213
K?, H?, 209, 268
lactate, H?, 212
lactose, H?, 212, 218220, 223
Na?,metabolite, 287, 288, 300
T
Taxis, 152, 196, 197, 251, 265
Tetrahydromethanopterin (MPT), 134
Tetraphenylborate, 345
Tetraphenylphosphonium, 345, 356,
358, 360
Thermogenesis, 122, 231, 232, 234, 236, 237,
240, 241, 246, 247
Thermophilic bacteria, 99, 285
Thiamine pyrophosphate, 18, 77
Thiosulfate, 131
Thylakoid, 24, 51, 55, 58, 60, 61, 65, 67, 68,
87, 106, 107, 131, 169, 210, 252,
253, 268, 269
Thymus involution, 360, 369, 375, 378
Thyroid hormones, 248, 324
Tonoplast, 178180, 184, 207, 289
Transducin, 154
Transhydrogenase, 11, 52, 187190, 299
Transmembrane difference in electric
potential (DW)
conversion into D pH, 249, 250, 268
definition, 9
in thylakoid, 67, 250, 268
in transmembrane protein transport, 226
Transmembrane difference in H?
concentration (DpH), 9
Subject Index
Transmembrane electrochemical potential
l H?)
difference in H? ions (D
as convertible energetic currency, 265
buffering, 265
definition, 207
transmission, 252, 253
Transmembrane electrochemical potential difl Na?), 12,
ference in Na? ions (D
135, 290
Tungsten, 282
U
Ubiquinone. See Coenzyme Q
UCP, 232236, 238, 239
Uncoupler, 131, 172, 196, 216, 219, 360, 366
Uncoupling
in brown fat, 232, 236, 238
in muscles, 237239
in plants, 239
Uniport of K?
in bacteria, 213
in chloroplasts, 269
in mitochondria, 269
Uveitis, 364
433
V
Valinomycin, 177, 181, 208, 218, 221, 287
Vanadate, 167, 180, 181, 282, 301
Vitamin A, 22, 309
Vitamin B1, 18
Vitamin B12, 18
Vitamin E, 344, 345
Vitamin R1, 53, 5658, 98
Vitamin R3, 96, 108, 247
W
Water-oxidizing complex (WOC), 57, 62
X
Xenobiotics, 126, 246, 247
Z
Zinc, 34, 108, 110
Index of Organisms
A
Acetobacterium woodii, 293
Acidaminococcus fermentans, 276
Acidithiobacillus (Thiobacillus) ferrooxidans,
129
Aplysia californica, 301
Arabidopsis thaliana, 330, 331
Arum sp., 240
Ascaris sp., 127, 128
Azotobacter vinelandii, 126, 128
B
Bacillus firmus, 291
Bacillus pseudofirmus FTU, 291
Bacillus sp. YN-1, 291
Bacillus subtilis, 196, 228
Bacillus thuringiensis, 301
Bdellovibrio bacteriovorus, 196
Blastochloris (Rhodopseudomonas) viridis,
3744, 4651
F
Florometra serratissima, 413, 414
Fusobacterium nucleatum, 276
H
Haemophilus influenzae, 228, 278
Halobacterium halobium, 139, 287
Halobacterium salinarium, 139, 141, 149, 151,
152, 251, 253, 267, 301
Helicobacter pylori, 96
Heterocephalus glaber, 340
Homo sapiens, 376, 377
I
Ilyobacter tartaricus, 164, 165, 176, 293
C
Caenorhabditis elegans, 337, 338
Candida albicans, 260
Ceriodaphnia affinis, 358, 368
Chara sp., 203
Chlamydomonas reinhardtii, 64
Chloroflexus sp., 55
Clostridium symbiosum, 276
L
Lactococcus casei, 159
Limonium vulgaris, 302
E
Endomyces magnusii, 260
Enterococcus hirae, 159, 180, 212,
282, 297
M
Malonomonas rubra, 277
Melipona bicolor, 413, 414
Methanobacterium thermoautotrophicum, 209
K
Klebsiella aerogenes, 275, 276
Klebsiella pneumoniae, 276, 278, 287
435
436
M (cont.)
Methanosarcina barkeri, 136
Methanosarcina mazei, 285
Mixotricha paradoxa, 204, 205
Moorella thermoacetica, 285
Mycobacterium phlei, 246, 287
N
Natronomonas pharaonis, 150
Neisseria gonorrhoeae, 278
Neisseria meningitidis, 278
Neurospora crassa, 94
Nitella sp., 203
Nitrobacter sp., 131
Nothobranchius furzeri, 335, 358, 368
Nothobranchius guentheri, 335
Nothobranchius kuhntae, 335
Nothobranchius rachovii, 335
Index of Organisms
R
Rhodospirillum rubrum, 35, 36, 51, 107, 184,
185, 187, 189, 190, 196, 268
Rickettsia prowazekii, 224
S
Saccharomyces cerevisiae, 166
Salinibacter ruber, 151
Salmonella typhimurium, 197, 276, 287, 297
Schizosaccharomyces pombe, 413
Spiroplasma sp., 203
Symplocarpus renifolius, 241
Synechococcus sp., 203, 204
Syntrophus gentianae, 186
T
Tenebrio molitor, 214
Thermotoga maritima, 285
Thermus thermophilus, 99, 101
O
Oncorhynchus keta, 329
P
Paracoccus denitrificans, 107, 110112, 123,
124, 126
Peptococcus aerogenes, 276
Phormidium uncinatum, 202, 203, 253, 267
Podospora anserina, 337, 358
Polytoma papillatum, 255
Polytomella agilis, 255
Porphyromonas gingivalis, 278
Propionigenium modestum, 276, 292, 293,
297, 300
Pseudomonas aeruginosa, 278
V
Veillonella alcalescens, 276
Vibrio alginolyticus, 277279, 287, 289291,
297, 300
Vibrio cholerae, 278, 291
Vibrio costicola, 277
Vibrio harveyi, 267, 278
Vibrio parahaemolyticus, 290
Y
Yarrowia lipolytica, 93, 101, 102
Yersinia pestis, 278